WO2023025173A1 - 特异性识别肺炎克雷伯菌o1抗原的抗体及其应用 - Google Patents

特异性识别肺炎克雷伯菌o1抗原的抗体及其应用 Download PDF

Info

Publication number
WO2023025173A1
WO2023025173A1 PCT/CN2022/114384 CN2022114384W WO2023025173A1 WO 2023025173 A1 WO2023025173 A1 WO 2023025173A1 CN 2022114384 W CN2022114384 W CN 2022114384W WO 2023025173 A1 WO2023025173 A1 WO 2023025173A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
seq
acid sequence
antibody
antigen
Prior art date
Application number
PCT/CN2022/114384
Other languages
English (en)
French (fr)
Inventor
牛玉强
晏丽
李忠
Original Assignee
舒泰神（北京）生物制药股份有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 舒泰神（北京）生物制药股份有限公司 filed Critical 舒泰神（北京）生物制药股份有限公司
Priority to CN202280003347.4A priority Critical patent/CN116033922A/zh
Publication of WO2023025173A1 publication Critical patent/WO2023025173A1/zh

Links

Images

Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • This application relates to an antibody or an antigen-binding fragment that specifically recognizes Klebsiella pneumoniae O1 antigen, its preparation method and use.
  • Klebsiella is a Gram-negative bacterium that is ubiquitous in nature. Klebsiella species can be present in the environment, such as surface water, sewage, soil, and plants, and can also colonize mucosal surfaces in mammals, such as humans, horses, or pigs (Matsen, J M et al. Applied microbiology vol.28,4(1974):672-8.; Bagley, S T. Infection control: IC vol.6,2(1985):52-8.; Rock, Clare et al. Infection control and hospital epidemiology vol. 35, 4(2014): 426-9.; Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4(1998): 589-603.).
  • Klebsiella can enter other tissues from mucosal surfaces and cause pneumonia, meningitis, liver abscess, urinary system inflammation, Diseases such as wound infection or sepsis (Tsay,Ren-Wen et al.Archives of internal medicine vol.162,9(2002):1021-7; Meatherall,Bonnie L et al.The American journal of medicine vol.122,9( 2009):866-73; Korvick, J A et al.Southern medical journal vol.84,2(1991):200-4.; Yu,Wen-Liang et al.Clinical infectious diseases: an official publication of the Infectious Diseases Society of America vol.42,10(2006):1351-8.; Ko, Wen-Chien et al.
  • Klebsiella infections are mainly caused by Klebsiella pneumoniae, the most medically important species of the genus Klebsiella (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), endotoxin (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, endotoxin (LPS) plays a very important role in the pathogenicity of bacteria, 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 lipid A
  • core oligosaccharide core oligosaccharide
  • O antigen O antigen
  • Prior art (such as international application publications WO2017064258A1, WO2018075375A1) also shows that antibodies against Klebsiella pneumoniae O1 antigen can not only kill or kill bacteria through opsonophagocytic killing (OPK) and/or complement-dependent killing (SBA), It can also neutralize the endotoxin in the blood and reduce the damage caused by Klebsiella pneumoniae to the body. It is therefore crucial to develop new anti-LPS antibodies to complement antibiotic therapy.
  • OPK opsonophagocytic killing
  • SBA complement-dependent killing
  • an isolated antibody specifically binding to Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:39 , 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 of SEQ ID NO:46.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or variants of said VH comprising up to about 5 amino acids in the HC-CDRs Substitution; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25 , and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 25 ID NO: 32, or a variant of said VL comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:39; and a V L comprising the amino acid sequence of SEQ ID NO:46 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:46 About 90% sequence identity.
  • an isolated antibody specifically binding to Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:40 , 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 of SEQ ID NO:47.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 8, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 14, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:40; and a V L comprising the amino acid sequence of SEQ ID NO:47 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:47 About 90% sequence identity.
  • an isolated antibody specifically binding to Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO: 41 , 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 of SEQ ID NO:48.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 9, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 15, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:20, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:27, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:34, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:41; and a V L comprising the amino acid sequence of SEQ ID NO:48 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:48 About 90% sequence identity.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:42 , 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 of SEQ ID NO:49.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 10, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 13, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:35, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:42; and a V L comprising the amino acid sequence of SEQ ID NO:49 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:49 About 90% sequence identity.
  • an isolated antibody specifically binding to Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:43 , 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 of SEQ ID NO:50.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 9, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 15, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:22, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:29, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any one of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprises: V H comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:43; and a V L comprising the amino acid sequence of SEQ ID NO:50 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:50 About 90% sequence identity.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:44 , 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 of SEQ ID NO:51.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 11, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:23, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:37, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:44; and a V L comprising the amino acid sequence of SEQ ID NO:51 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:51 About 90% sequence identity.
  • an isolated antibody specifically binding to Klebsiella pneumoniae O1 antigen comprising: V H comprising HC-CDR1 contained in V H as shown in the amino acid sequence of SEQ ID NO:45 , 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 of SEQ ID NO:52.
  • an isolated antibody specifically binding to the Klebsiella pneumoniae O1 antigen comprising: V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 , which comprises the amino acid sequence of SEQ ID NO: 12, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 17, or a variant of said VH comprising a substitution of up to about 5 amino acids in its HC-CDRs; and VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:38, or VL variants comprising up to about 5 amino acid substitutions in the LC-CDRs.
  • any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen comprise: V H comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant A body has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:45; and a V L comprising the amino acid sequence of SEQ ID NO:52 or a variant thereof having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO:52 About 90% sequence identity.
  • the isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen binds to the Klebsiella pneumoniae O1 antigen with a Kd value of 1 pM to 5 nM.
  • an isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen that binds Klebsiella pneumoniae competitively with any of the above-mentioned isolated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen.
  • Lebsiella In some embodiments, an isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is provided that specifically binds to the same epitope as any of the above-mentioned isolated antibodies that bind to the Klebsiella pneumoniae O1 antigen .
  • any of the isolated antibodies that specifically bind to a Klebsiella pneumoniae O1 antigen as described above, the isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen comprises an Fc fragment.
  • the isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is a full-length IgG antibody.
  • the isolated antibody that specifically binds the Klebsiella pneumoniae O1 antigen is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.
  • the isolated antibody that specifically binds the Klebsiella pneumoniae O1 antigen is chimeric, fully human or humanized.
  • the isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)' 2 , Fab'-SH , single chain antibody (scFv), Fv fragment, dAb, Fd, nanobody (nanobody), double chain antibody (diabody) and linear antibody.
  • an isolated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is provided, the antibody also binds to Klebsiella oxytoca, Klebsiella phytogenes in addition to the O1 antigen of Klebsiella pneumoniae.
  • an isolated nucleic acid molecule encoding an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen of any of the above.
  • 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 O1 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 O1 antigen comprising: a) culturing under conditions that can effectively express the antibody specifically binding to the Klebsiella pneumoniae O1 antigen any one of the above host cells; and b) obtaining the expressed antibody specifically binding to the Klebsiella pneumoniae O1 antigen from the host cells.
  • a method of treating, preventing or improving a disease or condition in a desired individual comprising administering to the individual an effective amount of any one of the above-mentioned compounds that specifically bind to the Klebsiella pneumoniae O1 antigen Antibody.
  • use of any one of the above-mentioned antibodies that specifically bind to Klebsiella pneumoniae O1 antigen in the preparation of a pharmaceutical composition for treating, preventing or improving a disease or condition in a desired individual is provided.
  • providing any one of the above-mentioned antibodies specifically binding to Klebsiella pneumoniae O1 antigen or a pharmaceutical composition comprising an antibody specifically binding to Klebsiella pneumoniae O1 antigen is prepared for treatment, Use in medicines for preventing or improving diseases or conditions.
  • the disease or disorder is related to Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris bacteria, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infection or colonization, including nosocomial infections, opportunistic infections, post-organ transplantation Infection and with Klebsiella, preferably Klebsiella serotype O1 (e.g.
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus other diseases or conditions associated with infection/colonization with Klebsiella granulomatosa, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis).
  • the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea, soft tissue infection, organ transplantation Post-infection, surgical infection, wound infection, pulmonary infection, suppurative liver abscess (PLA), lung abscess, cellulitis, necrotizing myositis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis , ankylosing spondylitis or spondyloarthropathies.
  • PHA suppurative liver abscess
  • lung abscess cellulitis, necrotizing myositis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis , ankylosing spondylitis or spondyloarthropathies.
  • compositions, kits and production products comprising any one of the above-mentioned antibodies specifically binding to Klebsiella pneumoniae O1 antigen.
  • FIGS 1A-1D are schematic diagrams of the structures of LPS of Klebsiella pneumoniae O1 and O2 serotypes.
  • the result shown in Figure 1A is a schematic diagram of the LPS structure of Klebsiella pneumoniae O1 serotype KP19173 strain;
  • the result shown in Figure 1B is a schematic diagram of the LPS structure of Klebsiella pneumoniae O1 serotype KP19213 strain;
  • Figure 1D is a schematic diagram of the LPS structure of the Klebsiella pneumoniae O2 serotype KP19203 strain.
  • FIGS. 2A-2B show that anti-O1 antibody G2 or G7 did not cross-react with Klebsiella pneumoniae O2 serotype.
  • the result shown in Figure 2A is that anti-O1 antibody G2 or G7 did not bind to LPS of Klebsiella pneumoniae O2 serotype KP19180 strain.
  • the result shown in Figure 2B is that the anti-O1 antibody G2 or G7 did not bind to the LPS of Klebsiella pneumoniae O2 serotype KP19203 strain.
  • Figures 4A-4B are the anti-O1 antibody G2 or G7 mediated serum bactericidal activity.
  • Figure 4A is the serum bactericidal activity mediated by anti-O1 antibody G2
  • Figure 4B is the serum bactericidal activity mediated by anti-O1 antibody G7.
  • FIGs 5A-5D show the binding of anti-O1 antibody G2 or G7 to LPS from other Klebsiella pneumoniae O1 serotypes.
  • the results shown in Figure 5A are the binding of anti-O1 antibody G2 or G7 to clinically isolated Klebsiella pneumoniae O1 serotype KP1953 strain LPS.
  • the results shown in Figure 5B are the binding of anti-O1 antibody G2 or G7 to clinically isolated Klebsiella pneumoniae O1 serotype KP1961 strain LPS.
  • the results shown in Figure 5C are the binding of anti-O1 antibody G2 or G7 to clinically isolated Klebsiella pneumoniae O1 serotype KP1962 strain LPS.
  • Figure 5D shows the results of the binding of anti-O1 antibody G2 or G7 to LPS of clinically isolated Klebsiella pneumoniae O1 serotype KP1963 strain.
  • the present application provides an antibody molecule (ie, an anti-O1 antibody) that specifically binds to the O1 antigen of Klebsiella pneumoniae.
  • an antibody molecule ie, an anti-O1 antibody
  • LPS neutralizing Klebsiella pneumoniae endotoxin
  • Anti-O1 antibodies provided by the present application include, for example, full-length anti-O1 antibodies, anti-Klebsiella pneumoniae O1 antigen single-chain antibodies (scFvs), anti-O1 Fc fusion proteins, multispecific (such as bispecific) anti-O1 antibodies , anti-O1 antibody immunoconjugates, and the like.
  • scFvs anti-Klebsiella pneumoniae O1 antigen single-chain antibodies
  • anti-O1 Fc fusion proteins anti-O1 Fc fusion proteins
  • multispecific (such as bispecific) anti-O1 antibodies anti-O1 antibody immunoconjugates, and the like.
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence DAWIS (SEQ ID NO:1); HC-CDR2 comprising the sequence RIRSKTDGETREYAAPVNG (SEQ ID NO:7); and HC-CDR3 comprising the sequence DPQWGI (SEQ ID NO:13); and a light chain variable domain (V L ), said V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASQSVSSRHLA (SEQ ID NO: 18); LC-CDR2 comprising the sequence GASSRAT (SEQ ID NO:25); and LC-CDR3 comprising the sequence QQYGSTPLT (SEQ ID NO:32).
  • V H comprises: a heavy chain complementarity determining region (HC
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence NAWMY (SEQ ID NO:2); HC-CDR2 comprising the sequence RIRSYSDGGTTDYAALVEG (SEQ ID NO:8); and HC-CDR3 comprising the sequence PSGDFYPAS (SEQ ID NO:14) and a light chain variable domain (V L ), said V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RSSQNLLHSNGYNYLD (SEQ ID NO: 19); LC-CDR2 comprising the sequence LSSNRAS (SEQ ID NO:26); and LC-CDR3 comprising the sequence MQALQTPYT (SEQ ID NO:33).
  • HC -CDR heavy chain complementarity
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence NFWMT (SEQ ID NO:3); HC-CDR2 comprising the sequence NINQGGTEGYYVDSVKG (SEQ ID NO:9); and HC-CDR3 comprising the sequence HHGWKYNSGWRTAFDI (SEQ ID NO:15); and a light chain variable domain (V L ), said V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASQGISNSLV (SEQ ID NO: 20); LC-CDR2 comprising the sequence GASKLHP (SEQ ID NO:27); and LC-CDR3 comprising the sequence QQSGRSPYT (SEQ ID NO:34).
  • HC -CDR heavy
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence DAWMS (SEQ ID NO:4); HC-CDR2 comprising the sequence RIRSKADGETIEYAAHVAG (SEQ ID NO:10); and HC-CDR3 comprising the sequence DPQWGI (SEQ ID NO:13); and a light chain variable domain ( VL ) comprising: light chain complementarity determining region (LC-CDR) 1 comprising the sequence KSSQSLLHSGGKTHFY (SEQ ID NO: 21); LC-CDR2 comprising the sequence EVSNRFS (SEQ ID NO:28); and LC-CDR3 comprising the sequence MQGTHWPPT (SEQ ID NO:35).
  • V H heavy chain complementarity determining region
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence NFWMT (SEQ ID NO:3); HC-CDR2 comprising the sequence NINQGGTEGYYVDSVKG (SEQ ID NO:9); and HC-CDR3 comprising the sequence HHGWKYNSGWRTAFDI (SEQ ID NO:15); and a light chain variable domain (V L ), said V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RTSQSISTHLN (SEQ ID NO: 22); LC-CDR2 comprising the sequence GASTLQN (SEQ ID NO:29); and LC-CDR3 comprising the sequence QQSYRIPYS (SEQ ID NO:36).
  • V H comprises: a
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC -CDR)1 comprising the sequence NYWMT (SEQ ID NO:5); HC-CDR2 comprising the sequence SINQGGSEQYYVDSLKG (SEQ ID NO:11); and HC-CDR3 comprising the sequence ADWMSIDH (SEQ ID NO:16); and a light chain variable domain (V L ), said V L comprising: light chain complementarity determining region (LC-CDR) 1 comprising the sequence RSSQSLVNSDGNIYLS (SEQ ID NO: 23); LC-CDR2 comprising the sequence QVSNRDS (SEQ ID NO:30); and LC-CDR3 comprising the sequence MQGTHWPWT (SEQ ID NO:37).
  • V H comprises: a heavy chain complementarity
  • the application provides an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen, the anti-O1 antibody comprising: a heavy chain variable domain (V H ), and the V H comprises: a heavy chain complementarity determining region (HC - CDR)1 comprising the sequence GYWMS (SEQ ID NO:6); HC-CDR2 comprising the sequence NIKQDGSEQYYVDSVKG (SEQ ID NO:12); and HC-CDR3 comprising the sequence DRGIKMGSVWYPSFDL (SEQ ID NO:17); and a light chain variable domain (V L ), said V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASRSISNYLN (SEQ ID NO: 24); LC-CDR2 comprising the sequence AASTLQS (SEQ ID NO:31); and LC-CDR3 comprising the sequence QQSYSAPRT (SEQ ID NO:38).
  • V H comprises: a
  • nucleic acids encoding anti-O1 antibodies are also provided.
  • compositions comprising anti-O1 antibodies are also provided.
  • treatment is a method of obtaining a beneficial or desired result, including a clinical result.
  • the beneficial or desired clinical result Including, but not limited to, one or more of the following: relieve one or more symptoms caused by the disease, reduce the extent of the disease, stabilize the disease (for example, prevent or delay the progression of the disease), prevent or delay the spread of the disease (for example, pathogenic bacteria systemic spread), preventing or delaying disease recurrence, delaying or slowing disease progression, improving disease state, remission of disease (partial or total), reducing dose of one or more other drugs needed to treat disease, delaying disease progression, improving or enhancing Quality of life, weight gain, and/or prolongation of survival.
  • treatment also includes the reduction of disease pathological outcomes (for example, for bacterial infections, bacterial load, inflammatory cell infiltration in tissue samples). The present application Methods contemplate any one or more aspects of these treatments.
  • prevention is meant to include preventive measures to prevent a disease or preventive measures to reduce the risk of a disease.
  • antibody includes full-length antibodies and antigen-binding fragments thereof.
  • 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.
  • the variable regions in both chains usually include 3 hypervariable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, heavy chain (HC ) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3).
  • CDRs complementarity determining regions
  • 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.
  • FRs flanking segments
  • 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.
  • major antibody classes are divided into subclasses such as IgG1 ( ⁇ 1 heavy chain), IgG2 ( ⁇ 2 heavy chain), IgG3 ( ⁇ 3 heavy chain), IgG4 ( ⁇ 4 heavy chain), IgA1 ( ⁇ 1 heavy chain) or IgA2 ( ⁇ 2 heavy chain).
  • antigen-binding fragment includes antibody fragments, for example, diabodies, Fab, Fab', F(ab') 2 , Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv) 2 , bispecific dsFv (dsFv-dsFv'), disulfide bond-stabilized diabody (ds diabody), single-chain antibody (scFv), scFv dimer (divalent diabody), Multispecific antibodies, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that are capable of binding to an antigen but do not contain the structure of a complete antibody, consisting of antibody fragments containing one or more CDRs .
  • 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).
  • Antigen-binding fragments also include fusion proteins comprising the antibody fragments described above.
  • 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.
  • epitope refers to a specific atom or group of amino acids on an antigen to which an antibody or antibody portion binds.
  • Epitopes may consist of carbohydrates, peptide structures, fatty acids, inorganic substances or their derivatives, organic substances, biochemical substances, or any combination thereof. If two antibodies or antibody portions appear to compete for binding to an antigen, they likely bind the same epitope on the antigen.
  • the primary antibody when the first antibody inhibits the binding of the second antibody to the Klebsiella pneumoniae O1 antigen target by at least 50% (eg, 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 O1 antigen target, and vice versa.
  • PCT publication WO 03/48731 describes a cross-competition-based high-throughput antibody "epitope-sorting" method.
  • the term “specifically binds”, “specifically recognizes” or “is specific for” refers to a measurable and reproducible interaction, for example binding of an antibody to a target 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 that is at least 10 times greater than its binding affinity for other targets.
  • an "isolated" anti-O1 antibody is an anti-O1 antibody that is (1) not related to a naturally occurring protein, (2) free of other proteins of the same origin, (3) produced from a different species expressed by cells of the genus, or (4) does not exist in nature.
  • isolated nucleic acid refers to nucleic acid of genomic, cDNA or synthetic origin or combinations thereof. According to its source, the "isolated nucleic acid” refers to (1) not related to all or part of the polynucleotide in the "isolated nucleic acid” found in nature, (2) may be associated with polynucleotides that are not associated with it in nature. The nucleotides are operably linked, or (3) do not occur in nature as part of a longer sequence.
  • CDR or "complementarity determining region” means the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides.
  • CDR or "complementarity determining region” means the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides.
  • 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. al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J.
  • 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 this (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 the 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 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 is a small antibody fragment prepared by constructing a scFv fragment (see the above paragraph) using a short linker (for example, 5 to 10 residues) between the V H and V L domains, This allows for interchain rather than intrachain pairing of the variable domains, 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 a non-human species such as mouse, rat, rabbit or Non-human mammalian hypervariable region residues with desired antibody specificity, affinity and performance are substituted (donor antibody).
  • HVR hypervariable region residues of the recipient antibody
  • donor antibody Non-human mammalian hypervariable region residues with desired antibody specificity, affinity and performance are substituted (donor antibody).
  • residues in the framework regions 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 all, 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 framework regions above 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
  • Percent amino acid sequence identity or “homology” of the polypeptide and antibody sequences identified herein is defined: a sequence comparison is made where conservative substitutions are considered to be part of the sequence identity, and the candidate sequence is Compare the percentage of identical amino acid residues in polypeptide sequences. 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.
  • percent amino acid sequence identity 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 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.
  • FcyRII receptors include FcyRIIA ("activating receptor") and FcyRIIB ("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.
  • the "CH1 domain" of the human IgG Fc 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 isotypes 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 the human IgG Fc region typically extends from amino acid 231 to amino acid 340.
  • the CH2 domain is unique in that it is not tightly paired with another region, but instead has two N-terminally linked branched sugar chains 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).
  • a "CH3" domain includes a stretch 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 for at least one FcR (e.g., a lower apparent Kd or IC50 value) 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
  • FcRs Fc receptors
  • NK cells only express 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 9:457-92 (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 of the antibody is substantially the same in number 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 herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).
  • Potency is a measure of the pharmacological activity of a compound expressed in terms of the amount required to produce an effect of a given intensity. It refers to the amount of compound required to achieve a defined biological effect; the smaller the dose required, the more effective the drug.
  • the potency of anti-O1 antibodies can be determined using, for example, the OPK assay or the SBA assay described herein.
  • OPK Opsonophagocytic killing
  • cell eg, Klebsiella pneumoniae
  • OPK activity detection are known in the art, see, for example, DiGiandomenico, A., et al., Infect Immun 72, 7012-7021 (2004).
  • SBA serum bactericidal assay
  • SBA also refers to the lysis of target cells by complement activation and formation of the "membrane attack complex" in the presence of complement.
  • Methods for assessing the efficacy of antibodies in killing target cells using the SBA assay are known in the art, see, e.g., Kobayashi, Scott D et al. "Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils.” mBio vol.9 , 2e00297-18.13 Mar. 2018.
  • Klebsiella infection is understood in the following way: Klebsiella is a Gram-negative bacterium which is a member of the family Enterobacteriaceae. It is a ubiquitous bacterium that can also colonize human hosts, usually in the gut or upper respiratory tract. As an opportunistic pathogen, it can invade germ-free sites from these sites without adequate control by the immune system. Uncontrolled bacterial replication at these sites induces inflammation that is largely mediated by endotoxin (LPS) molecules released from Klebsiella bacteria. In the case of bacteremia, endotoxin molecules can trigger septic shock.
  • LPS endotoxin
  • Klebsiella colonization means that a subject has a sufficiently high concentration of Klebsiella at a detectable site, but the bacteria do not cause any signs or symptoms. Colonization can persist for a long period of time and is influenced by the body's immune response to the organism, competition from other organisms, and sometimes the use of antimicrobial agents resulting in lysis (disintegration).
  • neutralization refers broadly to any molecule that inhibits a pathogen from exerting its biological activity, regardless of the mechanism by which neutralization is achieved, such as inhibition of Klebsiella (eg Klebsiella pneumoniae) Infect the host, or inhibit the pathogen to promote infection by producing endotoxin, or inhibit endotoxin.
  • neutralization can be achieved by antibodies that inhibit the colonization of host mucosal surfaces by Klebsiella (e.g., Klebsiella pneumoniae), invasion of sterile internal sites, and induction of undesirable biological signals (in the worst case, induction of septic shock) .
  • neutralization refers to inhibiting the binding of specific LPS to its cognate receptor (eg, Toll-like receptor-4 complex), thereby inhibiting its biological activity.
  • Such neutralizing potency is generally determined using standard assays known in the art, for example using in vitro or in vivo neutralization assays, such as the LAL assay or a TLR-4 reporter cell line. In these tests, for example, the inhibition of endotoxic biological activities is determined using colorimetric methods.
  • Antibodies against or neutralizing Klebsiella interfere with pathogens and pathogenic responses to be able to limit or prevent infection, and/or improve disease conditions caused by such infections, or inhibit Klebsiella pathogenesis (e.g. Klebsiella pneumoniae), especially to inhibit dissemination and replication of Klebsiella (e.g. Klebsiella pneumoniae) into or within a sterile body cavity/body part of a host dissemination and reproduction.
  • neutralizing antibodies can also be understood as "protective antibodies", referring to antibodies that induce the body's immune response to pathogenic agents observed in active or passive immunization.
  • the neutralizing or protective antibodies described in the present invention can be used for therapeutic purposes, for example for prophylaxis or therapy, ie preventing, ameliorating, treating or at least partially inhibiting disease symptoms, side effects or progression induced by pathogens.
  • Protective antibodies are capable of killing or preventing the replication of live Klebsiella (e.g. Klebsiella pneumoniae) cells following therapeutic application (i.e. administration on an existing infection), by e.g. inducing serum bactericidal or opsonophagocytic activity, or from The sterile in vivo site clears all bacterial cells or their LPS molecules.
  • Protective antibodies administered prophylactically may prevent the onset of infection (eg, prevent the spread of Klebsiella pneumoniae from a non-sterile site to a sterile body cavity) by the mechanisms mentioned above or by other mechanisms.
  • 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 identity 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 anti-O1 antibody or composition disclosed herein 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.
  • terapéuticaally effective amount refers to the amount or activity of an anti-O1 antibody disclosed herein or a composition thereof sufficient to cause beneficial effects or desired results (including clinical results) when administered to a subject.
  • an effective amount or synonym depends on the context in which it is administered.
  • An effective amount is intended to mean an amount of antibody or composition thereof sufficient to treat, prevent or inhibit such diseases or conditions.
  • a therapeutically effective amount of an antibody as described herein is particularly useful for treating, modulating, attenuating, reversing or affecting a disease or disorder that would benefit from the inhibition of Klebsiella (e.g.
  • Klebsiella pneumoniae bacteria pathogenesis, such as adhesion and colonization of mucosal surfaces, uncontrolled replication within the site of the bacterium, and toxicity of bacterial products to host cells.
  • an anti-O1 antibody or composition disclosed herein is capable of inhibiting the growth of Klebsiella (e.g., Klebsiella pneumoniae) and/or killing Klebsiella (e.g., Klebsiella pneumoniae), the anti-O1 antibody can is 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, and Does not cause significant adverse biological reactions, or interact 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 application described herein are to 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 cancer, meaning that the method is usually used to treat other types of cancer than type X cancer.
  • the present application provides an anti-O1 antibody that specifically binds to the O1 antigen of Klebsiella pneumoniae.
  • the anti-O1 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and/or light chain CDRs described herein.
  • the application provides an isolated antibody that binds to Klebsiella pneumoniae O1 serotype.
  • Contemplated anti-O1 antibodies include, for example, full-length anti-O1 antibodies (e.g., full-length IgG1 or IgG4), anti-O1 single-chain antibodies, anti-O1 Fc fusion proteins, multispecific (e.g., bispecific) anti-O1 antibodies, anti-O1 antibodies - immunoconjugates, and the like.
  • the anti-O1 antibody is a full-length antibody (eg, full-length IgG1 or IgG4) or an antigen-binding fragment thereof that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the anti-O1 antibody is Fab, Fab', F(ab)' 2 , Fab'-SH, single chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, diabody (diabody) or linear antibody.
  • the antibody that specifically binds to the Klebsiella pneumoniae O1 antigen refers to that the binding affinity of the antibody to the Klebsiella pneumoniae O1 antigen is at least 10 times that of the non-target binding affinity (including, for example, 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 times).
  • the anti-O1 antibody binds to Klebsiella pneumoniae O1 antigen, Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinky nose and/or Klebsiella rhinosclerosis O1 antigen.
  • non-target refers to an antigen that is not Klebsiella 01.
  • 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).
  • non-human anti-O1 antibodies comprising human sequences (eg, human heavy and light chain variable domains comprising human CDR sequences) are broadly discussed herein, non-human anti-O1 antibodies are also contemplated.
  • the non-human anti-O1 antibody comprises the human CDR sequences and non-human framework region sequences of the anti-O1 antibodies described herein, and in some embodiments, the non-human framework region sequences comprise any of the human CDR sequences for use as described herein.
  • non-human anti-O1 antibodies include anti-O1 antibodies produced by grafting one or more human CDR sequences described herein into non-human framework regions (eg, murine or chicken framework region sequences).
  • the anti-O1 antibodies described herein specifically recognize the Klebsiella pneumoniae O1 antigen.
  • the anti-O1 antibody is effective against Klebsiella (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinolyticum granulomatosis, Klebsiella stinknasia, and/or Klebsiella rhinosclerosis) O1 serotype specific and does not cross with other genera or non-O1 serotypes of Klebsiella reaction.
  • Klebsiella such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinolyticum granulomatosis, Klebsiella stinknasia, and/or Klebsiella rhinosclerosis
  • any anti-O1 antibody described herein comprising an antibody heavy chain constant region and an antibody light chain constant region.
  • the anti-O1 antibody comprises an IgG1 heavy chain constant region.
  • the anti-O1 antibody comprises an IgG2 type heavy chain constant region.
  • the anti-O1 antibody comprises an IgG3 type heavy chain constant region.
  • the anti-O1 antibody comprises an IgG4 type heavy chain constant region.
  • the heavy chain constant region comprises (including consists of or consists essentially of) the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises (including consists of or consists essentially of) the amino acid sequence of SEQ ID NO:54.
  • the anti-O1 antibody comprises a kappa light chain constant region.
  • the light chain constant region comprises (including consists of or consists essentially of) the amino acid sequence of SEQ ID NO:55.
  • the anti-O1 antibody comprises a lambda light chain constant region.
  • the light chain constant region comprises (including consists of or consists essentially of) the amino acid sequence of SEQ ID NO:56.
  • the anti-O1 antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or variants of said VH comprising up to about 5 amino acid substitutions in its HC-CDRs; and VL comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 32, or a variant of said VL , It contains up to about 5 amino acid substitutions in its LC-CDRs.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and V L comprising: LC- CDR1 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprising the amino acid sequence of SEQ ID NO :25, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:32.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:39.
  • the anti- O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:46.
  • the anti-O1 antibody comprises: 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: 39; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 comprised by the VL as shown in the amino acid sequence of SEQ ID NO:46.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%); and V L comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, said variant Has at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:46.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:39, and VL comprising the amino acid sequence of SEQ ID NO:46.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33, or a variant of said V L whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:33.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:40.
  • the anti- O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:47.
  • the anti-O1 antibody comprises: 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: 40; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:47.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, the Such variants have at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:47.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:40, and VL comprising the amino acid sequence of SEQ ID NO:47.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 34, or variants of said VL , whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:34.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:41.
  • the anti- O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:48.
  • the anti-O1 antibody comprises: 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: 41; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:48.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 90%, such as at least 91%, of the amino acid sequence of SEQ ID NO: 41 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof, said A variant has at least about 90%, eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:41, and VL comprising the amino acid sequence of SEQ ID NO:48.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35, or variants of said VL , whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:13; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:35.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:42.
  • the anti- O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:49.
  • the anti-O1 antibody comprises: 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: 42; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:49.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and VL , which comprises the amino acid sequence of SEQ ID NO: 49 or a variant thereof, wherein Such variants have at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:49.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:42, and VL comprising the amino acid sequence of SEQ ID NO:49.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 22, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36, or a variant of said V L whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:36.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:43.
  • the anti-O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:50.
  • the anti-O1 antibody comprises: 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: 43; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:50.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof, the Such variants have at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:50.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:43, and VL comprising the amino acid sequence of SEQ ID NO:50.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 23, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37, or a variant of said V L whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 16; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 23, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 30, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:37.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:44.
  • the anti-O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:51.
  • the anti-O1 antibody comprises: 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: 44; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:51.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof, the Such variants have at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:51.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:44, and VL comprising the amino acid sequence of SEQ ID NO:51.
  • the anti-O1 antibody comprises: VH
  • the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or variants of said VH comprising up to about 5 amino acid substitutions in the HC-CDRs; and VL comprising: LC-CDR1, which Comprising the amino acid sequence of SEQ ID NO: 24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38, or variants of said V L , whose LC- CDRs contain up to about 5 amino acid substitutions.
  • the anti-O1 antibody comprises: VH , and the VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:17; and V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:24, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:31, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:38.
  • the anti-O1 antibody comprises: VH comprising 1, 2 or 3 HC-CDRs in the amino acid sequence of SEQ ID NO:45.
  • the anti- O1 antibody comprises: V L comprising 1, 2 or 3 LC-CDRs in the amino acid sequence of SEQ ID NO:52.
  • the anti-O1 antibody comprises: 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: 45; and V L comprising Comprising LC-CDR1, LC-CDR2 and LC-CDR3 contained in the VL as shown in the amino acid sequence of SEQ ID NO:52.
  • the anti-O1 antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 45, or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity; and V L comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof, the Such variants have at least about 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the amino acid sequence of SEQ ID NO:52.
  • the anti-O1 antibody comprises: VH comprising the amino acid sequence of SEQ ID NO:45, and VL comprising the amino acid sequence of SEQ ID NO:52.
  • amino acid substitutions described above are limited to the "exemplary substitutions” shown in Table 4 herein. In some embodiments, amino acid substitutions are limited to the "preferred substitutions” shown in Table 4 herein.
  • the application provides antibodies that compete with any of the anti-O1 antibodies described herein for binding to the Klebsiella pneumoniae O1 antigen.
  • antibodies are provided that are capable of binding to Klebsiella pneumoniae O1 antigen competitively with any of the anti-O1 antibodies described herein.
  • anti-O1 antibodies are provided that bind to the same epitope as an anti-O1 antibody molecule comprising a VH and a VL , wherein the VH comprises amino acids set forth in any one of SEQ ID NOs: 39-45 sequence, and the V L comprises the amino acid sequence shown in any of SEQ ID NOs:46-52.
  • anti-O1 antibodies are provided that compete for binding to the Klebsiella pneumoniae O1 antigen with an anti-O1 antibody comprising a VH and a VL , wherein the VH comprises any of SEQ ID NOs: 39-45 an amino acid sequence shown, and said V L comprises any one of the amino acid sequences shown in SEQ ID NOs: 46-52.
  • competition assays can be used to identify monoclonal antibodies that compete with the anti-O1 antibodies described herein for binding to the Klebsiella pneumoniae O1 antigen. 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 the antibodies 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.
  • antibodies that compete with the anti-O1 antibodies described herein are chimeric, humanized, or fully human antibodies.
  • Exemplary anti-O1 antibody sequences are shown in Table 2 and Table 3, 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 antibodies as described herein, but based on prediction algorithms other than those exemplified in the table below.
  • Klebsiella infections are associated with hospitalization.
  • Klebsiella primarily attack immunocompromised individuals with severe underlying diseases such as diabetes or chronic pulmonary obstruction.
  • Botan, Bascomb and in Classify Klebsiella into 5 categories including Klebsiella pneumoniae, Klebsiella oxytogenes, Klebsiella terrestris, Klebsiella phytogenes and Klebsiella ornithinolyticus, and Klebsiella pneumoniae Also included are Klebsiella stinkpneumoniae and Klebsiella pneumoniae subsp.
  • 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 is a major surface antigen constructed from O-specific polysaccharides (O-PS), core oligosaccharides, and lipid A containing varying numbers of oligosaccharide repeating units (RU).
  • O-PS O-specific polysaccharides
  • RU oligosaccharide repeating units
  • O1 and O2 serotype pathogens account for 50-68% of all Klebsiella infections (Hansen, D S et al. Journal of clinical microbiology vol.37,1(1999):56 -62.; Follador, Rainer et al. Microbial genomics vol.2,8e000073.25 Aug.2016;).
  • the LPS expressed by O1 and O2 strains contained O-PS composed of galactans (gal) homopolymer.
  • O1 serotype expresses D-galactan-I (gal-I) composed of ⁇ 3)- ⁇ -D-Galf-(1 ⁇ 3)- ⁇ -D-Galp-(1 ⁇ as repeating units and antigen D-galactan-II (gal-II) consisting of ⁇ 3)- ⁇ -D-Galp-(1 ⁇ 3)- ⁇ -D-Galp-(1 ⁇ as a repeating unit (Whitfield, C et al.Journal of bacteriology vol.173,4(1991):1420-31.; Kol,O et al.Carbohydrate research vol.236(1992):339-44.).
  • Gal-II is unique to O1 type LPS (Pennini, Principle E et al. Nature communications vol.8,1 1991.8Dec.2017).
  • O2 is only composed of gal-I (Whitfield, C et al. Journal of bacteriology vol.174,15( 1992):4913-9.).
  • the synthesis of gal-I is encoded by the his-linked rfb (wb) operon (Clarke, B R, and C Whitfield.
  • gal-II can combine with gal-I or gal-III homopolymers, resulting in both cases for the O1 serotype.
  • the O2 serotype consists only of gal-I or gal-III homopolymers (Stojkovic, Katarina et al. Frontiers in microbiology vol.8 684.25 Apr.2017).
  • the anti-O1 antibody is a full length anti-O1 antibody.
  • the full length anti-O1 antibody is IgA, IgD, IgE, IgG or IgM.
  • the full-length anti-O1 antibody comprises an IgG constant region, such as the constant region of IgG1, IgG2, IgG3, IgG4, or variants thereof.
  • the full length anti-O1 antibody comprises a lambda light chain constant region.
  • the full length anti-O1 antibody comprises a kappa light chain constant region.
  • the full-length anti-O1 antibody is a full-length human anti-O1 antibody.
  • the full length anti-O1 antibody comprises a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-O1 antibody comprises an altered or otherwise altered Fc sequence such that it has altered or enhanced antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent Effector functions of cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent Effector functions of cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • a full-length anti-O1 antibody comprising an IgG1 constant region that specifically binds the Klebsiella pneumoniae O1 antigen.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG2 constant region, the anti-O1 antibody specifically binding to the Klebsiella pneumoniae O1 antigen.
  • the IgG2 is human IgG2.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG3 constant region, the anti-O1 antibody specifically binding to the Klebsiella pneumoniae O1 antigen.
  • the IgG3 is human IgG3.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region, said anti-O1 antibody specifically binding to Klebsiella pneumoniae O1 antigen.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; HC-CDR2, which Comprising the amino acid sequence shown in any one of SEQ ID NOs: 7-12 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and HC-CDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 13-17, or a variant thereof, said variant comprising at most about 3 to at most about 3 (eg 1, 2 or 3) amino acid substitutions; and b) light Chain variable structural domain, described light chain variable structural domain comprises: LC-CDR1, it comprises the aminoacid sequence shown in any one of SEQ ID NOs: 1-6 or
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG2 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; HC-CDR2, which Comprising the amino acid sequence shown in any one of SEQ ID NOs: 7-12 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and HC-CDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 13-17, or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and b) light chain variable structure domain, the light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence shown in any of SEQ ID NOs: 18-24 or
  • the IgG2 is human IgG2.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG3 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6 or a variant thereof comprising a substitution of at most about 3 (eg 1, 2 or 3) amino acids, HC-CDR2, which Comprising the amino acid sequence shown in any one of SEQ ID NOs: 7-12 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and HC-CDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 13-17, or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and b) light chain variable structure domain, the light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence shown in any of SEQ ID NOs: 18-24 or
  • the IgG3 is human IgG3.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs: 1-6 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; HC-CDR2, which Comprising the amino acid sequence shown in any one of SEQ ID NOs: 7-12 or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and HC-CDR3 comprising The amino acid sequence shown in any one of SEQ ID NOs: 13-17, or a variant thereof, said variant comprising at most about 3 (eg 1, 2 or 3) amino acid substitutions; and b) light chain variable structure domain, the light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence shown in any of SEQ ID NOs: 18-24 or
  • LC-CDR2 which comprises the amino acid sequence shown in any one of SEQ ID NOs: 25-31 or a variant thereof comprising up to about 3 (for example 1, 2 or 3) amino acid substitutions
  • LC-CDR3 comprising the amino acid sequence shown in any of SEQ ID NOs: 32-38 or a variant thereof comprising up to about 3 (eg 1, 2 or 3) Amino acid substitutions.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs:1-6, HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:7-12, and HC-CDR3, which comprises SEQ ID The amino acid sequence shown in any of NOs: 13-17, or a variant of the heavy chain variable domain, comprising at most about 5 (eg 1, 2, 3, 4 or 5) in its HC-CDR sequence ) amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs: 18-24, LC-CDR2 , which comprises the amino acid sequence shown in any of SEQ ID NOs:25-31, and LC-CDR3, which comprises the amino acid sequence shown in any of
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises a) a heavy chain variable domain comprising: HC-CDR1, It comprises the amino acid sequence shown in any one of SEQ ID NOs:1-6, HC-CDR2, it comprises the amino acid sequence shown in any one of SEQ ID NOs:7-12, and HC-CDR3, it comprises SEQ ID NOs : the amino acid sequence shown in any one of 13-17, or a variant of said heavy chain variable domain comprising at most about 5 (eg 1, 2, 3, 4 or 5) in its HC-CDR sequence Amino acid substitutions; and b) a light chain variable domain comprising: LC-CDR1 comprising the amino acid sequence shown in any of SEQ ID NOs: 18-24, LC-CDR2, It comprises the amino acid sequence shown in any of SEQ ID NOs:25-31, and LC-CDR3, it comprises the amino acid sequence shown in any of SEQ ID NOs
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs:1-6, HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:7-12, and HC-CDR3, which comprises SEQ ID The amino acid sequence shown in any of NOs:13-17; and b) the light chain variable domain comprising: LC-CDR1 comprising any of SEQ ID NOs:18-24
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence shown in any one of SEQ ID NOs:1-6, HC-CDR2, which comprises the amino acid sequence shown in any one of SEQ ID NOs:7-12, and HC-CDR3, which comprises SEQ ID The amino acid sequence shown in any of NOs:13-17; and b) the light chain variable domain comprising: LC-CDR1 comprising any of SEQ ID NOs:18-24
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain 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: 7, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 13; and b) a light chain variable domain
  • the The light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 18, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 25, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 32 .
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:8, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:14; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO :33.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO:15; and b) a light chain variable domain , the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence SEQ ID NO: 20, LC-CDR2, which comprises the amino acid sequence SEQ ID NO: 27, and LC-CDR3, which comprises the amino acid sequence SEQ ID NO: 34.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:4, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:10, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:13; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 21, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 28, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :35.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:9, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:15; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:22, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:29, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :36.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:5, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:11, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:16; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:23, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:30, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :37.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO: 6, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 12, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:24, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:31, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :38.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain 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: 7, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 13; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 18, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 25, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :32.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:2, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:8, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:14; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO :33.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:9, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:15; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 20, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 27, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :34.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:4, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:10, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:13; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 21, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 28, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :35.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:3, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:9, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:15; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:22, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:29, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :36.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO:5, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO:11, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:16; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:23, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:30, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :37.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: HC-CDR1 , which comprises the amino acid sequence of SEQ ID NO: 6, HC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 12, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain
  • the light chain variable domain comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:24, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:31, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO :38.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region wherein the anti-O1 antibody comprises: a heavy chain variable domain ( VH ), said VH comprising SEQ ID NOs: 39-45 Any of the amino acid sequences shown, or variants thereof, which have at least about 90% (e.g., at least 91%, 92%, 93%, 94%) of any of the amino acid sequences shown in SEQ ID NOs: 39-45 %, 95%, 96%, 97%, 98% or 99%) sequence identity; and a light chain variable domain (V L ), said V L comprising any of SEQ ID NOs:46-52 shown An amino acid sequence or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any of SEQ ID NOs: 46-52 , 96%, 97%, 98% or 99%) sequence identity.
  • VH heavy chain variable domain
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG2 constant region
  • the anti-O1 antibody comprises: a heavy chain variable domain ( VH ), said VH comprising SEQ ID NOs: 39-45 Any of the amino acid sequences shown, or variants thereof, which have at least about 90% (e.g., at least 91%, 92%, 93%, 94%) of any of the amino acid sequences shown in SEQ ID NOs: 39-45 %, 95%, 96%, 97%, 98% or 99%) sequence identity; and a light chain variable domain (V L ), said V L comprising any of SEQ ID NOs:46-52 shown An amino acid sequence or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any of SEQ ID NOs: 46-52 , 96%, 97%, 98% or 99%) sequence identity.
  • VH heavy chain variable domain
  • the IgG2 is human IgG2.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG3 constant region
  • the anti-O1 antibody comprises: a heavy chain variable domain ( VH ), said VH comprising SEQ ID NOs: 39-45 Any of the amino acid sequences shown, or variants thereof, which have at least about 90% (e.g., at least 91%, 92%, 93%, 94%) of any of the amino acid sequences shown in SEQ ID NOs: 39-45 %, 95%, 96%, 97%, 98% or 99%) sequence identity; and a light chain variable domain (V L ), said V L comprising any of SEQ ID NOs:46-52 shown An amino acid sequence or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any of SEQ ID NOs: 46-52 , 96%, 97%, 98% or 99%) sequence identity.
  • VH heavy chain variable domain
  • the IgG3 is human IgG3.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a heavy chain variable domain ( VH ), said VH comprising SEQ ID NOs: 39-45 Any of the amino acid sequences shown, or variants thereof, which have at least about 90% (e.g., at least 91%, 92%, 93%, 94%) of any of the amino acid sequences shown in SEQ ID NOs: 39-45 %, 95%, 96%, 97%, 98% or 99%) sequence identity; and a light chain variable domain (V L ), said V L comprising any of SEQ ID NOs:46-52 shown An amino acid sequence or a variant thereof having at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any of SEQ ID NOs: 46-52 , 96%, 97%, 98% or 99%) sequence identity.
  • VH heavy chain variable domain
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region comprising an IgG1 constant region, wherein the anti-O1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 39-45 , and a light chain variable domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 46-52.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: a heavy chain variable domain comprising the amino acid sequence shown in any one of SEQ ID NOs:39-45 , and a light chain variable domain comprising the amino acid sequence shown in any one of SEQ ID NOs: 46-52.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:39; and a V L comprising the amino acid sequence of SEQ ID NO:46 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:46 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:40; and a V L comprising the amino acid sequence of SEQ ID NO:47 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:47 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a V L comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 48 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant Having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 42; and a V L comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 49 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:43; and a V L comprising the amino acid sequence of SEQ ID NO:50 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:50 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:44; and a V L comprising the amino acid sequence of SEQ ID NO:51 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:51 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG1 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 45; and a V L comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 52 have at least about 90% sequence identity.
  • the IgG1 is human IgG1.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53 and the light 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:53 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:39; and a V L comprising the amino acid sequence of SEQ ID NO:46 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:46 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:40; and a V L comprising the amino acid sequence of SEQ ID NO:47 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:47 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a V L comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 48 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region wherein the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant Having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 42; and a V L comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 49 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:43; and a V L comprising the amino acid sequence of SEQ ID NO:50 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:50 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:44; and a V L comprising the amino acid sequence of SEQ ID NO:51 or a variant thereof which is identical to the amino acid sequence of SEQ ID NO:51 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • a full-length anti-O1 antibody comprising an IgG4 constant region
  • the anti-O1 antibody comprises: VH , said VH comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 45; and a V L comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof that is identical to the amino acid sequence of SEQ ID NO: 52 have at least about 90% sequence identity.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:55. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54 and the light 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:54 and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:56.
  • Binding affinity is expressed as Kd, Koff, Kon or Ka.
  • Koff refers to the rate constant for dissociation of an antibody from an antigen/antibody complex, as determined by a kinetic selective device.
  • Kon refers to the association rate constant for the binding of an antibody to an antigen to form an antigen/antibody complex.
  • the equilibrium dissociation constant Kd used herein 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 sites and reaches equilibrium in the antibody molecule solution, which is equal to Koff /Kon. The determination of Kd assumes that all bound molecules are in solution.
  • the corresponding equilibrium 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 dissociation constant (Kd) can be used as an indicator of the affinity of the antibody moiety for the antigen.
  • 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 anti-O1 antibody specifically binds to the Klebsiella pneumoniae O1 antigen target with 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 anti-O1 antibody 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 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10
  • the Kd value for the binding of the anti-O1 antibody to the non-target is higher than the Kd value of the anti-O1 antibody to the target, and in some embodiments cited herein, the anti-O1 antibody binds to the target (e.g. The binding affinity of anti-O1 antibodies to non-targets was higher.
  • a non-target refers to an antigen other than the Klebsiella pneumoniae O1 antigen.
  • the Kd value of the anti-O1 antibody (for the Klebsiella pneumoniae O1 antigen) binding to the non-Klebsiella pneumoniae O1 antigen target is at least 10 times different, such as 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 non-target binding Kd value of the anti-O1 antibody is 10 ⁇ 1 M to 10 ⁇ 6 M (for example, 10 ⁇ 1 M to 10 ⁇ 6 M, 10 ⁇ 1 M to 10 ⁇ 5 M, 10 -2 M to 10 -4 M).
  • the non-target refers to an antigen other than the Klebsiella pneumoniae O1 antigen.
  • the Kd value for binding between the anti-O1 antibody and the non-Klebsiella pneumoniae O1 antigen target is 10 ⁇ 1 M to 10 ⁇ 6 M, 1 ⁇ 10 ⁇ 1 M to 5 ⁇ 10 ⁇ 6 M, 10 -1 M to 10 -5 M, 1 ⁇ 10 -1 M to 5 ⁇ 10 -5 M, 10 -1 M to 10 -4 M, 1 ⁇ 10 -1 M to 5 ⁇ 10 -4 M, 10 -1 M to 10 -3 M, 1 ⁇ 10 -1 M to 5 ⁇ 10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M , 1 ⁇ 10 -2 M to 5 ⁇ 10 -6 M, 10 -2 M to 10 -5 M, 1 ⁇ 10 -2 M to 5 ⁇ 10 -5 M, 10 -2 M to 10 -4 M , 1 ⁇ 10 -2 M to 5 ⁇ 10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1 ⁇ 10 -3 M to 5 ⁇ 10 -3 M to 5 ⁇ 10
  • the anti-O1 antibody when referring to an anti-O1 antibody that specifically recognizes a Klebsiella pneumoniae O1 antigen target with a high binding affinity and binds a non-target with a low binding affinity, the anti-O1 antibody binds to Klebsiella pneumoniae
  • the Kd value of O1 antigen target binding is 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, 10 -10 M to 10 -12 M)
  • the Kd value for non-target binding is 10 -1 M to 10 -6 M (for example, 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).
  • the binding affinity of the anti-O1 antibody is compared to that of a control anti-O1 antibody (e.g., MPG196) or other anti-O antigen antibodies (for example, the binding affinities of KPN70 or G3-78) were compared.
  • the Kd value of the combination between the control anti-O1 antibody or the anti-O antigen antibody and the Klebsiella pneumoniae O1 antigen can be the combination between the anti-O1 antibody described in the present application and the Klebsiella pneumoniae O1 antigen. At least 2 times the Kd value, such as 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10-100 times, 100-1000 times, 10 3 -10 4 times.
  • nucleic acid molecules encoding anti-O1 antibodies are also contemplated.
  • a nucleic acid (or set) encoding a full-length anti-O1 antibody is provided, including any full-length anti-O1 antibody described herein.
  • the nucleic acid (or a set of nucleic acids) of the anti-O1 antibody described herein may also include a nucleic acid sequence encoding a polypeptide tag (eg, protein purification tag, His tag, HA tag).
  • an isolated host cell comprising an anti-O1 antibody, an isolated nucleic acid encoding an anti-O1 antibody polypeptide component, or a vector comprising a nucleic acid encoding an anti-O1 antibody polypeptide component described herein.
  • a variant includes a nucleotide sequence that hybridizes to a nucleic acid sequence encoding an anti-O1 antibody of the present 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 anti-O1 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, promoters (e.g., lymphocyte-specific promoter) and 3' untranslated region (UTR), can express anti-O1 antibody (such as full-length anti-O1 antibody).
  • promoters e.g., lymphocyte-specific promoter
  • UTR 3' untranslated region
  • 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 begins to decline when 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 , MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, for example including but not limited to actin promoter, myosin promoter, Hemoglobin promoter and creatine kinase promoter.
  • SV40 Simian virus 40
  • MMTV mouse mammary tumor virus
  • HV-LTR human immunodeficiency virus long terminal repeat
  • MoMuLV promoter avian leukemia virus promoter
  • Epstein-Barr virus immediate early promoter Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter Rous sarcoma virus promoter
  • human gene promoter for example including but not limited to actin
  • 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.
  • the expression of anti-O1 antibodies is inducible.
  • the nucleic acid sequence encoding the anti-O1 antibody is operably linked to an inducible promoter, including any of the inducible promoters 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 anti-O1 antibody is the Tet system. In some embodiments, the inducible promoter system used to express the anti-O1 antibody is the E. coli lac repression 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 transcriptional activation domain.
  • a change of 4 amino acids in the DNA-binding region of TetR altered the binding properties of rtTA so 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 (see, 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 that exhibits 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 full-length anti-O1 antibodies described herein is provided.
  • the nucleic acid comprises one or more nucleic acid sequences encoding full-length anti-O1 antibody heavy and light chains.
  • 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.
  • the vector can 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. Examples include Southern and Northern blotting, RT-PCR, and PCR; "biochemical” assays, such as detection of the presence or absence of a specific polypeptide, e.g. identified by immunological methods (ELISAs and Western blots) or by assays described herein All fall within the scope of this application.
  • the anti-O1 antibody is or is derived from a monoclonal antibody.
  • the anti-O1 antibody comprises VH and VL from a monoclonal antibody, or a variant thereof.
  • the anti-O1 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, phage display methods, or using recombinant DNA methods. Additionally, exemplary phage display methods are described herein and in the Examples below.
  • lymphocytes In the hybridoma cell method, hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce 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 lines fuse efficiently, ensure high-level stable expression of antibodies by the selected antibody-producing cells, and are sensitive to certain media, such as HAT media.
  • 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.
  • the anti-O1 antibody comprises a sequence selected from a clone of an antibody library, eg, a phage library displaying scFv or Fab fragments.
  • a clone of an antibody library eg, a phage library displaying scFv or Fab fragments.
  • Such clones can be identified by screening a combinatorial library of antibody fragments having the desired activity.
  • various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding properties. These methods are reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001), and in, for example, McCafferty et al.
  • 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 anti-O1 antibody is prepared by phage display screening the anti-O1 antibody part in the library that can specifically bind to the target Klebsiella pneumoniae 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 to the target Klebsiella pneumoniae O1 antigen can be screened by iterative binding of the phage to the target Klebsiella pneumoniae O1 antigen bound to a solid support (such as beads for solution panning 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.
  • suitable host cells such as E.coli XL1-Blue
  • Multiple rounds of panning e.g., 2, 3, 4, 5, 6 or more rounds
  • solution panning cell panning, or a combination of the two
  • Phage cloning The specific binding of the enriched phage clones to the target Klebsiella pneumoniae O1 antigen can be detected by any method known in the art, including, for example, ELISA and FACS.
  • Another way to screen antibody libraries is to display proteins on the surface of yeast cells.
  • Wittrup et al. (US Patents 6,699,658 and 6,696,251) developed a method for displaying libraries in yeast cells.
  • one component includes the yeast lectin protein (Aga1) anchored on the yeast cell wall, and the other component includes the second subunit of the lectin protein Aga2, which can Combined with Aga1 protein and displayed on the surface of yeast cells.
  • the Agal protein is expressed by integrating the Agal gene into the yeast chromosome. After transformation of a single-chain variable fragment (scFv) library fused to the Aga2 gene in a yeast display plasmid, the library is retained in yeast due to the presence of an additional nutritional marker. Both Aga1 and Aga2 proteins are expressed under the control of a galactose-inducible promoter.
  • scFv single-chain variable fragment
  • VH and VK fragments Human antibody V gene repertoires ( VH and VK fragments) were obtained by the PCR method using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998).
  • PCR templates were derived from commercially available RNA or cDNA, including PBMC, spleen, lymph node, bone marrow and tonsil. After combining independent VH and VK PCR libraries, they were assembled into scFv format by overlap extension PCR (Sheets, MD et al, Proc. Natl. Acad. Sci. USA 95, 6157-6162 1998).
  • yeast scFv display library To construct a yeast scFv display library, the resulting scFv PCR product was cloned into a yeast display plasmid in yeast by homologous recombination. (Chao, G, et al, Nat Protoc. 2006; 1(2):755-68. Miller KD, et al. Current Protocols in Cytometry 4.7.1-4.7.30, 2008).
  • Anti-O1 antibodies can be screened using a mammalian cell display system, in which the antibody is partially displayed on the cell surface and the antibody specifically targeting the Klebsiella pneumoniae O1 antigen is isolated by an antigen-directed screening method (such as U.S. patent No. 7,732,195 described in B2).
  • a library of Chinese hamster ovary (CHO) cells displaying a large number of human IgG antibody genes can be created and used to discover clones expressing high-affinity antibody genes.
  • An alternative display system has been developed that enables simultaneous cell surface display and secretion of the same protein through alternative splicing, where the displayed protein phenotype remains genotype-related, enabling simultaneous biophysical and cellular function-based analysis The secreted soluble antibody was characterized in .
  • This method overcomes many limitations of previous mammalian cell displays and enables direct screening and maturation of antibodies in the form of full-length, glycosylated IgGs (Peter M. Bowers, et al, Methods 2014, 65:44-56).
  • Transient expression systems are suitable for single rounds of antigen selection prior to antibody gene restoration and are therefore most useful for selecting antibodies from smaller libraries.
  • Stable exosome vectors offer an attractive option. Exosome vectors can be efficiently transfected and stably maintained at low copy numbers, allowing multiple rounds of panning and resolution of more complex antibody repertoires.
  • the IgG library is constructed based on the ligation of germline sequence V gene segments isolated from a panel of human donors with rearranged (D)J regions. RNA collected from human blood samples was reverse transcribed into cDNA, V H and V K fragments were amplified using V H and V K specific primers, and purified by gel extraction. IgG libraries were prepared by subcloning VH and VK fragments into display vectors containing IgG1 or K constant regions, respectively, and then electroporating or transducing 293T into cells.
  • VH and VK were ligated to generate scFv, which was then subcloned into a display vector, which was then electroporated or transduced into 293T cells.
  • IgG libraries are constructed based on germline sequence V gene fragments and rearranged (D)J regions isolated from a group of donors, such as mice, rats, rabbits or monkeys.
  • 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 Klebsiella pneumoniae O1 antigen-specific yeast clone 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 can also be modified, for example by replacing human heavy and light chain constant regions with coding sequences and/or by replacing homologous non-human sequences with framework regions (U.S. Patent No. 4,816,567; Morrison et al., supra), or by A coding sequence of a non-immunoglobulin polypeptide covalently linked to all or part of a coding sequence of an immunoglobulin.
  • This non-immunoglobulin polypeptide can replace the constant region of the antibody of
  • 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.
  • Antibody variable domains 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 regions.
  • the first heavy chain constant region (CH1) 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.
  • the anti-O1 antibody (such as a full-length anti-O1 antibody) can be a fully human antibody or a humanized 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. In some embodiments, 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 in which 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 domain.
  • 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.
  • Fully human antibodies are 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
  • human germline immunoglobulin gene arrays into such germline mutant mice produces human antibodies upon antigen stimulation, see, e.g., 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; and WO 97/17852.
  • 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).
  • Fully human antibodies 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 amino acid sequences of anti-O1 antibody variants provided herein are also contemplated.
  • the amino acid sequences of antibody variants can be prepared by introducing appropriate modifications into the antibody-encoding nucleotide sequence or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the antibody amino acid sequence.
  • 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.
  • anti-O1 antibody variants 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 antibody of interest and the product screened for desired activity, for example, improved biological activity, retained/improved antigen binding ability, reduced immunogenicity, or improved ADCC or CDC.
  • 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, the variant antibody portions are displayed on phage, and the variants are screened for specific biological activities (eg, based on neutralizing activity or binding affinity). Alterations (eg, substitutions) in regions of the HVRs can be made to achieve improved neutralizing activity or binding affinity. Alterations can be made in HVR "hot spots", 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.
  • a useful method for identifying amino acid residues or regions of an antibody that can be targeted for mutation 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
  • 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 antibodies with a methionyl residue at the N-terminus.
  • Other insertional variants of antibody molecules include fusions to the N-terminus or C-terminus of the antibody molecule of an enzyme (eg, ADEPT) or a polypeptide that increases the serum half-life of the antibody.
  • one or more amino acid modifications are introduced into the Fc region of an antibody described herein (eg, a full-length anti-O1 antibody or an anti-O1 antibody fusion protein), resulting in an Fc region variant.
  • the Fc region variants have enhanced ADCC, CDC and/or ADCP potency, generally associated with Fc-binding receptors (FcRs).
  • the Fc region variant has reduced ADCC, CDC and/or ADCP potency.
  • Antibodies can be modified and/or altered in the Fc region to provide desired effector functions or serum half-life.
  • a naked antibody bound on the cell surface can induce cytotoxicity by: via antibody-dependent cellular cytotoxicity (ADCC); via recruit complement in complement-dependent cytotoxicity (CDC); or by recruiting non-specific cytotoxic cells expressing one or more effector ligands that recognize bound on bacteria such as Klebsiella pneumoniae Antibodies and subsequent phagocytosis of cells in antibody-dependent cell-mediated phagocytosis (ADCP); or by some other mechanism.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC recruit complement in complement-dependent cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • modified and/or altered Fc regions can be used, eg, to increase binding affinity for FcRn and to increase serum half-life.
  • the Fc region can also be conjugated to moieties such as PEG or albumin to increase serum half-life.
  • ADCC Antibody-dependent cell-mediated cytotoxicity
  • 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 anti-O1 antibody variants (e.g., full-length anti-O1 antibody variants) comprising an Fc region with partial but not all effector functions, such that they have an extended half-life in vivo, however specific For effector functions (such as CDC or ADCC) that are dispensable or deleterious, such anti-O1 antibodies are ideal candidates for this application.
  • anti-O1 antibody variants e.g., full-length anti-O1 antibody variants
  • Fc region with partial but not all effector functions such that they have an extended half-life in vivo, however specific For effector functions (such as CDC or ADCC) that are dispensable or deleterious, such anti-O1 antibodies are ideal candidates for this application.
  • Fc receptor (FcR) binding assay is used to confirm that the antibody lacks Fc ⁇ R binding (and thus likely lacks ADCC activity) but retains FcRn binding.
  • NK cells express only 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. 9:457-492 (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. USA 82:1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.
  • nonradioactive detection methods can be used (see, for example, ACTI TM Flow Cytometry Nonradioactive Cytotoxicity Assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TM Nonradioactive Cell Toxicity assays (Promega, Madison, Wis.). Effector cells used in such assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, ADCC activity of the molecule of interest is performed in vivo Assay, e.g., 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, for example, C1q and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, a CDC assay can be performed (see, for example, 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).
  • a variant of an anti-O1 antibody comprising a variant Fc region with one or more amino acid substitutions capable of enhancing ADCC effect.
  • the Fc region variant comprises one or more amino substitutions capable of enhancing the ADCC effect, and these substitutions are at positions 298, 333 and/or 334 (EU residue numbering) of the Fc region.
  • the anti-O1 antibody (eg, full-length anti-O1 antibody) variant comprises amino acid substitutions at positions S298A, E333A, and K334A in the Fc region.
  • alterations in the Fc region result in alterations (i.e., enhancement or attenuation) of Clq binding and/or complement-dependent cytotoxicity (CDC), see U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. al., J. Immunol. 164:4178-4184 (2000).
  • CDC complement-dependent cytotoxicity
  • an anti-O1 antibody eg, a full-length anti-O1 antibody
  • a variant of the Fc region with one or more amino acid substitutions capable of extending half-life or enhancing interaction with an Fc receptor (FcRn ) combination.
  • Antibodies with extended half-life and improved FcRn binding are described in US 2005/0014934A1 (Hinton et al.). These antibody Fc regions contain one or more amino acid substitutions that enhance the binding of the Fc region to FcRn.
  • 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).
  • anti-O1 antibodies eg, full-length anti-O1 antibodies
  • Fc variants described herein or combinations thereof.
  • an anti-O1 antibody provided herein is altered to increase or decrease the degree of glycosylation of the anti-NGF antibody.
  • Addition or deletion of glycosylation sites on the anti-O1 antibody can be conveniently achieved by changing the amino acid sequence of the anti-NGF antibody or its polypeptide portion to add or remove one or more glycosylation sites.
  • the sugar attached to it can be changed.
  • 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.
  • the anti-O1 antibodies of the present application can be modified with oligosaccharides to generate anti-O1 antibody variants with certain improved properties.
  • 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 FcRIIIa 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.
  • anti-O1 antibody e.g., full-length anti-O1 antibody
  • variants comprise an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may ADCC function will be enhanced.
  • anti-O1 antibodies that have reduced fucose relative to the same anti-O1 antibodies produced by wild-type CHO cells. That is, they 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).
  • the N-linked glycans of the anti-O1 antibody have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose.
  • the fucose content of the anti-O1 antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%.
  • the N-linked glycans of the anti-O1 antibody do not comprise fucose, i.e., wherein the anti-O1 antibody is completely fucose-free, or fucose-free, or afucosylated.
  • 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 2004/056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as ⁇ -1,6-fucosyltransferase gene, FUT8 gene Knockout 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).
  • Anti-O1 antibody (eg, full-length anti-O1 antibody) variants further provide bisected oligosaccharides, eg, wherein the biantennary oligosaccharide attached to the Fc region of the anti-O1 antibody is bisected by GlcNAc.
  • Such anti-O1 antibody (eg, full-length anti-O1 antibody) variants 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. 6,602,684 (Umana et al.); US 2005/0123546 (Umana et al.), and Ferrara et al.
  • anti-O1 antibody eg, full-length anti-O1 antibody
  • anti-O1 antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region.
  • Such anti-O1 antibody variants 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.).
  • the anti-O1 antibody (eg, full-length anti-O1 antibody) variant comprises an Fc region that binds Fc ⁇ RIII.
  • the anti-O1 antibody (e.g., full-length anti-O1 antibody) variant comprising an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or in combination with other antibodies having a human wild-type IgG1 Fc region. Enhanced ADCC activity in the presence of human effector cells compared to the same anti-O1 antibody (eg, full-length anti-O1 antibody).
  • cysteine-engineered anti-O1 antibody eg, a full-length anti-O1 antibody
  • the substituted residue occurs at a site accessible to the anti-O1 antibody.
  • a reactive sulfhydryl group located at an accessible site of the anti-O1 antibody can be used to conjugate the anti-O1 antibody to other moieties, such as drug moieties or linker-drug moieties, to prepare anti-Klebsiella pneumoniae O1 antigen immunoconjugates as further described herein.
  • Cysteine-engineered anti-O1 antibodies eg, full-length anti-O1 antibodies
  • the anti-O1 antibodies provided herein can be further modified to include other non-protein moieties known in the art and readily available.
  • Moieties suitable for derivatizing anti-O1 antibodies 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- vinylpyrrolidone) 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-dioxo
  • 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 anti-O1 antibody 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 or function of the anti-O1 antibody, whether anti-O1 antibody derivatives are useful for specific conditions treatment etc.
  • compositions comprising any of the anti-O1 antibodies (e.g., full-length anti-O1 antibodies), nucleic acids encoding the antibodies, vectors comprising nucleic acids encoding the antibodies, or host cells comprising the nucleic acids or vectors described herein. substances, also referred to herein as preparations).
  • a pharmaceutical composition comprising any anti-O1 antibody described herein and a pharmaceutically acceptable carrier.
  • a suitable anti-O1 antibody can be obtained by mixing an anti-O1 antibody of the desired purity with optionally a pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).
  • Antibody preparations are prepared in the form of lyophilized preparations or liquid preparations.
  • 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 lyophilized formulations can be reconstituted with a suitable diluent into a high protein concentration formulation, and the reconstituted formulation can be administered subcutaneously to the individual to be treated herein. Cationic liposomes or liposomes can be used to deliver the anti-O1 antibodies of the present application to cells.
  • the preparation described herein 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 .
  • other active substances necessary for the treatment of specific diseases, preferably substances with complementary activities and no adverse reactions to each other .
  • antibiotics steroidal and non-steroidal inflammation inhibitors
  • other antibacterial, anti-inflammatory agents are present in combination in amounts effective for the intended purpose. Effective amounts of other substances depend on the amount of anti-O1 antibody in the formulation, the type of disease or disorder or treatment, and other factors as described above. These agents are generally used at the same doses and routes of administration as described herein, or at 1% to 99% of the currently employed doses.
  • the anti-O1 antibody (e.g., full-length anti-O1 antibody) can also be embedded in microcapsules prepared, e.g., by coacervation techniques and interfacial polymerization, e.g., in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, respectively). , microemulsions, nanoparticles and nanocapsules) or in macroemulsions of hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules. Sustained release formulations can be prepared.
  • sustained release formulations of anti-O1 antibodies can be prepared.
  • suitable examples of 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 (U.S. Pat. No.
  • L-glutamine Acid and ethyl L-glutamate copolymer non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer such as LUPRON DEPOTTM (composed of lactic acid-glycolic acid copolymer and leuprolide acetate can 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 anti-O1 antibody (e.g., a full-length anti-O1 antibody) is formulated in the presence of citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or in any combination of the above buffers.
  • Preparations for in vivo administration must be sterile. This is readily accomplished, for example, by filtration using sterile filtration membranes.
  • Anti-O1 antibodies e.g., full-length anti-O1 antibodies
  • compositions described herein can be administered to individuals (e.g., mammals, such as humans) to treat or prevent infections with Klebsiella, preferably Klebsiella Bacteria O1 serotypes (e.g.
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosa, Klebsiella stinkana diseases and/or conditions associated with Klebsiella and/or Klebsiella rhinosclerosis) infection or colonization (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections) , Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinky nose and/or Klebsiella rhinosclerosis other diseases associated with bacterial) infection), including but not limited to pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea
  • the present application provides a treatment or prophylaxis with Klebsiella, preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes) , Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infection or conditions associated with infection or colonization ( For example, nosocomial infections, opportunistic infections, post-organ transplant infections, and infections with Klebsiella species (e.g.
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Ornithinolytica Klebsiella, Klebsiella granulomatosis, Klebsiella stinky nose and/or Klebsiella rhinosclerosis) infection-related methods
  • an anti-O1 antibody e.g. , a full-length anti-O1 antibody
  • a pharmaceutical composition such as any anti-O1 antibody (eg, a full-length anti-O1 antibody) described herein.
  • the individual is a human.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • Klebsiella terrestris Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinknasia, and/or Klebsiella rhinosclerosis
  • colonization-associated disease eg, nosocomial infection , opportunistic infections, post-transplant infections, and Klebsiella species (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus , Klebsiella granulomatosis, Klebsiella odoriferous nose and/or other diseases associated with Klebsiella rhinosclerosis) infection), comprising administering to said individual an effective amount of A pharmaceutical composition of an anti-O1 antibody (eg, a full-length anti-O1 antibody) to a Lebsiella O1 antigen.
  • an anti-O1 antibody eg,
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis, diarrhea, soft tissue infection, organ transplantation Post-infection, surgical infection, wound infection, pulmonary infection, suppurative liver abscess (PLA), lung abscess, cellulitis, necrotizing myositis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis , ankylosing spondylitis or spondyloarthropathies.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a pharmaceutical combination comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody) wherein said anti-O1 antibody comprises: V H , said V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising:
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 39; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:2, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:8, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 14, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 40 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 40; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 15, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 15, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising:
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 41; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH comprising: HC- CDR1 comprising the amino acid sequence of SEQ ID NO: 4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 13, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising: LC-C
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 42 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 42; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 15, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:3, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:9, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 15, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising:
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 43; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 16, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:5, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:11, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 16, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising:
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 44 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 44; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes
  • a composition comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 17, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising: VH , said VH comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:6, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:12, and HC-CDR3 , comprising the amino acid sequence of SEQ ID NO: 17, or a variant of said V H comprising up to about 5 amino acid substitutions in its HC-CDRs; and a V L comprising:
  • the anti-O1 antibody is a full length antibody. In some embodiments, the full length anti-O1 antibody is an IgG1 or IgG4 antibody.
  • the disease or condition is selected from, for example, 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 (PLA), lung abscess, cellulitis, necrotizing myocarditis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing Meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • the individual is a human.
  • a method for treating or preventing Klebsiella preferably Klebsiella serotype O1 (such as Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis) infection or colonization-associated disease (eg, nosocomial infection, opportunistic sexual infection, infection after organ transplantation, and other diseases associated with Klebsiella infection), comprising administering to said individual an effective amount of a composition comprising an anti-O1 antibody, wherein said antibody comprises: V H ,
  • the VH comprises the amino acid sequence of SEQ ID NO: 45 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 45; and a VL comprising the amino acid sequence of S
  • the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgGl or IgG4 constant region.
  • the IgG1 is human IgG1.
  • the IgG4 is human IgG4.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53.
  • the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54.
  • the light 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:56.
  • any one of the methods of treatment or prevention described herein is further provided with Klebsiella, preferably Klebsiella O1 serotype (such as Klebsiella pneumoniae, Klebsiella oxytoca phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose and/or Klebsiella rhinosclerosis) infection-related diseases and and/or therapeutic or prophylactic effects of a condition.
  • Klebsiella preferably Klebsiella O1 serotype (such as Klebsiella pneumoniae, Klebsiella oxytoca phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose and/or Klebsiella rhinosclerosis) infection-related diseases and and/or therapeutic or prophylactic effects of a condition.
  • the method prevents Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris , Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infection.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris , Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis
  • the individual is a mammal (eg, a human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, a clinical trial volunteer, an experimental animal, or the like. In some embodiments, the individual is less than 60 years old (including, for example, less than 50, 40, 30, 25, 20, 15, or 10 years old). In some embodiments, the individual is greater than 60 years old (including, for example, greater than 70, 80, 90, or 100 years old).
  • the individual is diagnosed with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella spp., Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinknasia, and/or Klebsiella rhinosclerosis) infection or immunocompromised, immunosuppressed susceptibility to Klebsiella , preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella spp., Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinknasia, and/or Klebsiella rhinosclerosis) infection
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis bacteria, Klebsiella odorifera and/or Klebsiella rhinosclerosis
  • infection such as nosocomial infection, opportunistic infection, infection after organ transplantation, and other diseases associated with Klebsiella infection
  • their contacts such as nosocomial infection, opportunistic infection, infection after organ transplantation, and other diseases associated with Klebsiella infection.
  • the individual is present with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris , Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infection.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris , Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxyto
  • the individual has a foreign body implanted, such as, but not limited to, a ventilator or catheter.
  • the individual is diagnosed with or is genetically predisposed to an immunodeficiency disorder, including but not limited to HIV infection, AIDS, and/or neutropenia.
  • the individual has received one or more forms of chemotherapy.
  • the individual has been treated with one or more forms of glucocorticoids.
  • the individual has received one or more forms of chemotherapy.
  • the individual is diagnosed with or is genetically predisposed to cancer, diabetes, and/or chronic structural lung disease (eg, cystic fibrosis or COPD).
  • the individual is diagnosed with or is genetically predisposed to a dysbiosis of the digestive system and/or other organs.
  • the individual has one or more risk factors associated with one or more of the diseases or conditions described above.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella spp., Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris Klebsiella spp., Klebsiella ornithinolyticus, Klebsiella granulomatosis, Klebsiella stinkhose, and/or Klebsiella rhinosclerosis
  • anti-O1 antibodies described herein e.g., full-length anti-O1 antibodies
  • compositions are combined with a second, third, or fourth agent (e.g., antibiotics, steroids, and non-steroidal inflammation inhibitors, and and/or other antibacterial or anti-inflammatory agents) in combination for the treatment or prophylaxis of Klebsiella, preferably Klebsiella serotype O1 (e.g.
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes , Klebsiella terrestris, Klebsiella ornithinolytica, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infection-related diseases.
  • the antibiotic is a penicillin, a cephalosporin, a carbapenem, a fluoroquinolone, an aminoglycoside, a monoamine, a polymyxin, An antibiotic composition containing a ⁇ -lactamase inhibitor, or any combination thereof.
  • the antibiotic is cefepime, ceftazidime, cefpirome, imipenem, meropenem, ticarcillin, piperacillin, azlocillin, carbenicillin, mezlocillin, atra Amikacin, tobramycin, gentamicin, amikacin, ciprofloxacin, levofloxacin, cefoperasone-sulbactam, piperacillin-tazobactam, fosfomycin, or any combination thereof.
  • the antibiotic is one or more of imipenem, tobramycin, ciprofloxacin, meropenem, or atritreonam.
  • the antibiotic is one or more of gentamicin, ampicillin or kanamycin.
  • compositions administered to an individual may vary depending on the particular composition, mode of administration, and type of disease being treated.
  • the amount of the composition e.g., a composition comprising an anti-O1 antibody
  • an objective response eg, partial response or complete response.
  • the amount of anti-O1 antibody composition is sufficient to produce a complete response in an individual. In some embodiments, the amount of anti-O1 antibody composition is sufficient to produce a partial response in an individual. In some embodiments, the anti-O1 antibody composition is administered at a dose (e.g., when administered alone) sufficient to produce greater than 20%, 25%, 30%, 35%, or more in a population of individuals treated with the anti-O1 antibody composition. 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% overall response rate.
  • An individual's response to the treatment methods described herein can be determined, for example, by detection of Klebsiella using Gram stain or other phenotypic testing methods.
  • the amount of the composition is sufficient to prolong progression-free survival in the individual. In some embodiments, the amount of the composition is sufficient to prolong the overall survival of the subject. In some embodiments, the amount of the composition (eg, when administered alone) is sufficient to produce greater than 50%, 60%, 70%, or 77% clinical benefit in a population of individuals treated with an anti-O1 antibody composition.
  • the amount of the composition is comparable to that before treatment with the same subject. It is sufficient to reduce Klebsiella, preferably Klebsiella serotype O1 (e.g. Klebsiella pneumoniae) organ burden to an intensity of at least 10 compared to or compared to the corresponding organ burden of other subjects not receiving treatment %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%.
  • Klebsiella serotype O1 e.g. Klebsiella pneumoniae
  • the magnitude of this therapeutic effect can be measured using standard methods, such as in vitro assays of purified enzymes, cell-based assays, animal models, or human trials.
  • the amount of anti-O1 antibody (e.g., full-length anti-O1 antibody) in the composition is lower than that which causes a toxic effect (i.e., a level of toxicity above a clinically acceptable level). effect), or at a level at which potential side effects can be managed or tolerated.
  • the amount of the composition approximates the maximum tolerated dose (MTD) of the composition. In some embodiments, the amount of the composition is greater than 80%, 90%, 95%, or 98% of the MTD.
  • the amount of anti-O1 antibody (eg, full-length anti-O1 antibody) in the composition ranges from 0.001 ⁇ g to 1000 ⁇ g.
  • the effective amount of the anti-O1 antibody (eg full-length anti-O1 antibody) in the composition is in the range of 0.1 ⁇ g/kg to 100 mg/kg based on body weight.
  • Anti-O1 antibody compositions can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous injection, intraarterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, Intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, mucosal or transdermal.
  • sustained release formulations of the compositions are used.
  • the composition is administered intravenously.
  • the composition is administered arterially.
  • the composition is administered intraperitoneally.
  • the composition is administered intrahepatically.
  • the composition is administered by hepatic artery infusion.
  • the composition is administered at a site remote from the first lesion.
  • an article of manufacture comprising a substance for use in the treatment or prevention of Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae) in an individual.
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae
  • 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 to treat or prevent a disease or condition described herein and has a sterile port (e.g., the container can be an IV bag or a vial with a hypodermic needle-pierceable cap) ).
  • At least one active substance in the composition is the anti-O1 antibody described in this application.
  • the label or package insert indicates that the composition is used to treat or prevent a particular condition.
  • the label or package insert further comprises instructions for administering the anti-O1 antibody composition to a patient. Articles of manufacture and kits comprising combination therapies are contemplated herein.
  • Package insert means the instructions normally included in commercial packages of therapeutic or prophylactic products, which contain information on the indications, usage, dosage, administration, contraindications and/or warnings relating to the use of such therapeutic or prophylactic products.
  • the package insert indicates that the composition can be used to treat or prevent diseases associated with Klebsiella, preferably Klebsiella serotype O1 infection (such as nosocomial infection, opportunistic infection, infection after organ transplantation, and with Klebsiella, preferably Klebsiella serotype O1 (e.g.
  • Klebsiella pneumoniae Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, granulomatosis, Klebsiella stinky nose and/or Klebsiella rhinosclerosis) infection).
  • the package insert indicates that the composition can be used to treat or prevent the following diseases, including pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis , diarrhea, soft tissue infection, post-transplant infection, surgical infection, wound infection, lung infection, suppurative liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis , meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy.
  • diseases including pneumonia, urinary tract infection, sepsis/bacteremia/sepsis, neonatal sepsis/bacteremia/sepsis , diarrhea, soft tissue infection, post-transplant infection, surgical infection, wound infection, lung infection, suppurative liver abscess (PLA), lung abscess, cellulitis, necrotizing
  • 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, for example for the treatment or prophylaxis of Klebsiella, preferably Klebsiella serotype O1 (e.g. Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes) Klebsiella terrestris, Klebsiella terrestris, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infections (e.g.
  • Klebsiella serotype O1 e.g. Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes) Klebsiella terrestris, Klebsiella terrestris, Klebsiella granulomatosis, Klebsiella stink-nasal and/or Klebsiella rhinosclerosis
  • Klebsiella serotype O1 e.g. Klebsiella
  • Kits of the present application include one or more containers comprising an anti-O1 antibody composition (or single dosage form and/or preparation), and in some embodiments, further comprising another agent (e.g., an agent described herein) ) and/or instructions for use consistent with any of the methods described herein.
  • the kit may further include instructions for selecting an individual suitable for treatment or prophylaxis.
  • 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 includes a composition comprising an anti-O1 antibody (eg, a full-length anti-O1 antibody).
  • the kit comprises: a) a composition comprising any one of the anti-O1 antibodies described herein, and b) at least one effective amount of another agent capable of enhancing the effect of the anti-O1 antibody (e.g., therapeutic effect, detection effect).
  • the kit comprises: a) a composition comprising any of the anti-O1 antibodies described herein, and b) administering the anti-O1 antibody composition to an individual for the treatment or prophylaxis of Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes, Klebsiella terrestris, Klebsiella ornithinolyticus, Klebsiella granulomatosis, Instructions for use of diseases associated with Klebsiella stink-nasal and/or Klebsiella rhinosclerosis) infections (such as nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with Klebsiella infection).
  • Klebsiella serotype O1 e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella phytogenes, Klebs
  • the kit comprises: a) a composition comprising any one of the anti-O1 antibodies described herein, and b) at least one effective amount of another agent capable of enhancing the effect of the anti-O1 antibody (e.g., therapeutic effect, detection of effect) and c) administering anti-O1 antibody compositions and other substances to individuals for the treatment or prevention of diseases associated with Klebsiella, preferably Klebsiella serotype O1 infection (e.g. nosocomial infections, opportunistic infections , infection after organ transplantation, and other diseases associated with Klebsiella infection).
  • the anti-O1 antibody and other substances may be present in separate containers or in the same container.
  • the kit may include one particular composition or two or more compositions, wherein one composition includes an anti-O1 antibody and the other composition includes another agent.
  • the kit comprises a nucleic acid (or set) encoding an anti-O1 antibody (eg, a full-length anti-O1 antibody).
  • the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody), and b) a host expressing the nucleic acid (or set of nucleic acids) cell.
  • the kit comprises: a) a (or set of) nucleic acid encoding an anti-O1 antibody (eg, a full-length anti-O1 antibody), and b) instructions for use in: i) in a host cell Expressing anti-O1 antibodies, ii) preparing a composition comprising anti-O1 antibodies, and iii) administering to an individual a composition comprising anti-O1 antibodies to treat or prevent infection associated with Klebsiella, preferably Klebsiella serotype O1 Diseases (eg, nosocomial infections, opportunistic infections, post-transplant infections, and other diseases associated with Klebsiella infection).
  • an anti-O1 antibody eg, a full-length anti-O1 antibody
  • the kit comprises: a) a (or set of) nucleic acid encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody), b) a host cell expressing the nucleic acid (or set of nucleic acids) , and c) instructions for use, suitable for: i) expressing the anti-O1 antibody in a host cell, ii) preparing a composition comprising the anti-O1 antibody, and iii) administering to an individual the composition comprising the anti-O1 antibody for the treatment or prevention of Klebsiella, preferably Klebsiella serotype O1 infection-associated diseases (eg nosocomial infections, opportunistic infections, post-organ transplant infections and other diseases associated with Klebsiella infections).
  • an anti-O1 antibody e.g., a full-length anti-O1 antibody
  • a host cell expressing the nucleic acid (or set of nucleic acids)
  • instructions for use suitable for:
  • 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. Accordingly, the present application also provides articles of manufacture including vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like.
  • the instructions for use of the anti-O1 antibody composition usually include some information, such as dosage, administration cycle and administration route, etc.
  • the container can be unit dose, bulk (eg, multi-dose package) or subunit dose.
  • a kit comprising a sufficient dose of an anti-O1 antibody as described herein (e.g., a full-length anti-O1 antibody) is provided for long-term effective treatment of an individual, e.g., 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 time.
  • the kit can also comprise multiple unit doses of the anti-O1 antibody, the pharmaceutical composition and instructions for use, and be packaged in quantities sufficient for storage and use in pharmacies, eg, hospital pharmacies and compounding pharmacies.
  • KP19173 represents the Klebsiella pneumoniae O1 serotype whose LPS contains D-gal I and D-gal II repeating units (Fig. 1A); and KP19213 represents the LPS containing D-gal III and Klebsiella pneumoniae O1 serotype with two types of repeating units of D-galII (Fig. 1B); KP19180 representing LPS containing D-gal I repeating unit O2 serotype of Klebsiella pneumoniae (Fig. 1C); representing LPS with KP19203 Klebsiella pneumoniae O2 serotype containing the D-gal III repeat unit (Fig. 1D).
  • the anti-O2 antibody KPN70 disclosed in the patent application CN109689090A binding to the D-gal I repeating unit
  • the anti-O antigen antibody G3-78 disclosed in the patent application CN107371365A binding to the D-gal III repeating unit
  • the anti-O1 antibody MPG196 disclosed in patent application WO2018/029356A1 binding D-gal II repeating unit
  • Embodiment 1 Prepare LPS, O-PS and screen anti-O1 single-chain antibody (scFv)
  • the pellet was completely resuspended with 70% ethanol, centrifuged at 13000 rpm and 4°C for 15 min, and the supernatant was discarded. Place the centrifuge tube in an ultra-clean bench and dry it to obtain the crude extract of LPS.
  • the crude extract was configured into a 2% (w/v) solution, an equal amount of 95% ethanol was added, and stirred while adding. Centrifuge at 12000rpm for 30min to remove the precipitate (repeated twice). Take the supernatant and place it in a new tube, add 6 times the volume of 95% ethanol, and at the same time add sodium acetate with a final concentration of 0.1%.
  • Phenol-sulfuric acid Determination of polysaccharide content by colorimetry [C].//Proceedings of Chinese Pharmaceutical Association National Biochemical New Drug Research and Clinical Application Academic Conference. Qingdao: Chinese Pharmaceutical Association Biochemical and Biotechnology Drug Professional Committee, 2004:176-178.).
  • scFv single-chain antibodies
  • 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.
  • V L and V H were amplified from yeast expression vectors and constructed into eukaryotic expression vectors pTT5-L (containing kappa constant region) and pTT5-H1 (containing IgG1 heavy chain constant region) or pTT5-H4 (containing IgG4 heavy 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 with Protein A affinity chromatography. Briefly, the protein A column was first equilibrated with 6 column volumes of 50 mM PBS buffer (containing 0.15M 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.
  • KPN70, G3-78 and MPG196 were set as control antibodies.
  • LPS was dissolved in PBS solution (adjusted pH to 7.2, final concentration was 0.2 ⁇ g/mL), coated 96-well plate at 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.
  • the results shown in Table 5 show that the anti-O1 antibodies G1-G7 antibodies and the control antibody MPG196 can bind to both KP19173 strain LPS and KP19213 strain LPS. Compared with the control antibody MPG196, the ability of the anti-O1 antibody G1-G7 antibodies to bind KP19173 strain or KP19213 strain LPS was better or equivalent.
  • the control antibody KPN70 can only bind to KP19173 strain LPS, and the control antibody G3-78 can only bind to KP19213 strain LPS.
  • 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, resulting in the activation of downstream NF- ⁇ B transcription factors and thereby secretion of SEAP, as measured by QUANTI-Blue TM substrate turnover.
  • HEK-Blue TM hTLR4 cells were cultured according to the manufacturer's instructions, and seeded into 96-well culture plates with about 2.5 ⁇ 10 4 cells per well; after that, 10 ⁇ L of LPS prepared in Example 1 ( 10 ⁇ g/mL, derived from KP19173 strain or KP19213 strain) and the initial concentration of 1mg/mL anti-O1 antibody (reconstituted into human IgG1 form) serial dilutions were pre-mixed, added to a 96-well culture plate, 37 ° C, 5% CO 2 Incubate for 6-16 hours.
  • the anti-O1 antibody G1-G7 antibody and the control antibody MPG196 had neutralizing activity against KP19173 strain and KP19213 strain LPS.
  • the neutralization activity of the anti-O1 antibody G1-G7 antibodies against KP19173 strain or KP19213 strain LPS was better or equivalent.
  • the control antibody KPN70 only had neutralizing activity against KP19173 strain LPS; the control antibody G3-78 had neutralizing activity only against KP19213 strain LPS.
  • the anti-O1 antibody G1-G7 antibody neutralizes KP19173 strain LPS with comparable effect.
  • the anti-O1 antibody G1-G7 antibody showed a comparable effect of neutralizing KP19213 strain LPS.
  • Biacore 3000 was used to characterize the binding affinities of full-length anti-O1 antibodies G2 and G7 (reconstructed into human IgG1 form) and control antibodies MPG196 and KPN70 to Klebsiella pneumoniae O-PS.
  • the Bio-OPS prepared in Example 1 (from KP19173 strain or KP19213 strain) was coated on the surface of a streptavidin chip (streptavidin chip), and the affinity of antibodies at different concentrations to Bio-OPS was detected.
  • the antibody with an initial concentration of 50 ⁇ g/mL was diluted 1:4 to obtain 7 concentration gradients.
  • the binding time was set to 120 s
  • the dissociation time was set to 80 s
  • the flow rate was 30 ⁇ L/min.
  • the on-rate and off-rate of the antibody are measured using SPR technique and the binding affinity is determined.
  • the anti-O1 antibody G2 or G7 has a high binding affinity to KP19173 strain or KP19213 strain O-PS.
  • the affinity of G7 to Klebsiella pneumoniae KP19173 strain or KP19213 strain O-PS was increased by nearly 10 times, and compared with the control antibody KPN70, the affinity was increased by nearly 50 times and 600 times, respectively.
  • the affinity of anti-O1 antibody G2 to K. pneumoniae KP19173 strain or KP19213 strain O-PS was comparable to that of the control antibody MPG196, and nearly 2 times and 20 times that of the control antibody KPN70, respectively.
  • Klebsiella pneumoniae serotype O1 and O2 serotype O antigens contain the same galactan repeating unit, which is prone to cross-reaction.
  • the following assay was designed to detect the specificity of anti-O1 antibody binding to Klebsiella pneumoniae O1 serotype: KP19180 and KP19203 are two strains of Klebsiella pneumoniae O2 serotype, which represent O2 sera containing different galactose repeat units in OPS type strains.
  • the bactericidal activity mediated by full-length anti-O1 antibody G2 or G7 (reconstituted into human IgG1 form) and control antibody MPG196, G3-78 was determined by opsonophagocytic killing (OPK) assay.
  • OPK opsonophagocytic killing
  • the plasmid pUC18-mini-Tn7T-Gm-lux expressing complete luciferin was electrotransformed into Klebsiella pneumoniae (KP19173 strain) to construct luminescent Klebsiella pneumoniae.
  • the killing activity mediated by each antibody was determined by detecting relative light units (RLU) of luminescent Klebsiella pneumoniae.
  • OPK OPK oxidative stress protein kinase
  • MPG196 which binds to the Gal-II epitope and thus can bind to KP19173
  • G3-78 which can bind to the Gal-III epitope and therefore cannot bind to KP19173 can be used as a negative control antibody.
  • luminescent Klebsiella pneumoniae KP19173 strain
  • diluted baby rabbit serum Cedarlane, 1:10
  • macrophages differentiated from HL-60 macrophages differentiated from HL-60
  • different concentrations of antibodies were mixed in a 96-well plate Mix and incubate for 2h at 37°C with shaking (250rpm).
  • Use a microplate reader to measure the value of relative light units. Set the relative light unit values measured without adding antibodies and Klebsiella pneumoniae as 0% and 100% killing rates, respectively, to calculate the anti-O1 antibodies G2, G7 and control antibodies MPG196, G3 at different concentrations
  • the relative killing rate of -78 was drawn using Graphpad Prism software to determine the opsonophagocytosis killing activity of each antibody.
  • the complement-dependent bactericidal activity mediated by the full-length anti-O1 antibody G2 or G7 (reconstructed into human IgG1 form) and the control antibody KPN70 was determined by serum bactericidal assay (SBA) test.
  • SBA serum bactericidal assay
  • the plasmid pUC18-mini-Tn7T-Gm-lux expressing complete luciferin was electrotransformed into Klebsiella pneumoniae KP19173 strain to construct luminescent Klebsiella pneumoniae.
  • Complement-dependent bactericidal activity mediated by anti-O1 antibodies G2, G7 (reconstituted into human IgG1 form) and control antibody KPN70 was determined by measuring relative light units (RLU) of luminescent Klebsiella pneumoniae.
  • the detection method of SBA is as described in the literature (for example, see Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils, mBio. 2018 Mar-Apr; 9(2):e00297-18.). Briefly, the luminescent Klebsiella pneumoniae KP19173 strain and antibody gradient dilutions with an initial concentration of 1.3 nM were pre-mixed, added to a 96-well culture plate, and incubated at 37°C, 5% CO 2 for 15 min. After adding 25 ⁇ L of diluted baby rabbit serum and 35 ⁇ L of PBS buffer, incubate for 3 h at 37° C. with shaking (250 rpm).
  • Relative light units were then measured using a microplate reader.
  • the relative light unit values obtained when no antibody and Klebsiella pneumoniae were not added were set as 0% and 100% killing rate, respectively, to calculate the anti-O1 antibody G2, G7 and control antibody KPN70 at different concentrations.
  • the relative killing rate was drawn using Graphpad Prism software to determine the serum bactericidal activity of each antibody.
  • the ELISA binding test described in Example 2 was used to detect the binding situation of the anti-O1 antibodies G2, G7 and LPS from other clinically isolated Klebsiella pneumoniae O1 serotype strains (KP1953, KP1961, KP1962 or KP1963 strains), To determine whether the anti-O1 antibody G2 or G7 has broad-spectrum binding to Klebsiella pneumoniae O1 serotype strains.
  • Example 7 Effect of anti-O1 antibody in mouse bacteremia model
  • mice were sensitized to endotoxin by intraperitoneal injection of 20 mg D-(+)-galactosamine hydrochloride (GalN) (sigma, G1639-5G), and then 100 ⁇ L bacterial solution (KP19173 or KP19213 strain) was injected through the tail vein to Build a bacteremia model. If all mice die within 24 hours after injection, it means that the modeling is successful, and this dose is the lethal dose, and this dose is used as the modeling dose in subsequent experiments.
  • GalN D-(+)-galactosamine hydrochloride
  • anti-O1 antibody G2 or G7 has a protective effect
  • 24 hours before the establishment of the mouse model different concentrations of anti-O1 antibody G2, G7 (reconstituted into human IgG1 form), positive control antibody MPG196 or negative control antibody were injected intraperitoneally.
  • Control antibody HIV antibody HIV-10E8 Broad and potent neutralization of HIV-1 by a gp41-specific human antibody Nature 491(7424), 406-412(2012)).
  • anti-O1 antibody G2 or G7 can improve the survival rate of mice, and its protective effect is also Comparable to control antibody MPG196 (results not shown).
  • the anti-O1 antibody G2 or G7 can significantly improve the survival rate of mice, and has a preventive and protective effect.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Mycology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段，及其制备方法和用途。

Description

特异性识别肺炎克雷伯菌O1抗原的抗体及其应用
相关申请的交叉引用
本申请要求申请号为202110980272.2，申请日为2021.08.25，发明名称为“特异性识别肺炎克雷伯菌O1抗原的抗体及其应用”的中国专利申请的优先权，且该申请的全部内容以引用方式并入本文中。
对电子序列表的引用
电子序列表(文本名称：CN_202107212436_SEQLIST.xml，记录日期：2022.07.12，大小：51KB)的内容通过整体引用并入本文中。
技术领域
本申请涉及特异性识别肺炎克雷伯菌O1抗原的抗体或抗原结合片段，及其制备方法和用途。
背景技术
克雷伯(氏)菌属是一种革兰氏阴性菌，其在自然界中无处不在。克雷伯氏菌属可以存在于环境中，例如地表水、污水、土壤和植物上，也可以定植于哺乳动物的粘膜表面，如人类、马、或猪(Matsen,J M et al.Applied microbiology vol.28,4(1974):672-8.；Bagley,S T.Infection control:IC vol.6,2(1985):52-8.；Rock,Clare et al.Infection control and hospital epidemiology vol.35,4(2014):426-9.；Podschun,R,and U Ullmann.Clinical microbiology reviews vol.11,4(1998):589-603.)。对于免疫力降低/受损、免疫缺陷或长期大量使用抗生素导致菌群失调的人群来说，克雷伯氏菌会从粘膜表面进入其他组织并引起肺炎、脑膜炎、肝脓肿、泌尿***炎症、伤口感染或败血症等疾病(Tsay,Ren-Wen et al.Archives of internal medicine vol.162,9(2002):1021-7；Meatherall,Bonnie L et al.The American journal of medicine vol.122,9(2009):866-73；Korvick,J A et al.Southern medical journal vol.84,2(1991):200-4.；Yu,Wen-Liang et al.Clinical infectious diseases:an official publication of the Infectious Diseases Society of America vol.42,10(2006):1351-8.；Ko,Wen-Chien et al.Emerging infectious diseases vol.8,2(2002):160-6.)。克雷伯氏菌感染主要由肺炎克雷伯菌引起的，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)、内毒素(lipopolysaccharide，LPS)、黏附因子、铁载体***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。其中内毒素(LPS)在细菌的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的结构有相当大的变化，但其通常由脂质 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)。由于O抗原的变化远小于CPS，因此有学者认为克雷伯氏菌属LPS的O抗原可以作为免疫治疗的潜在靶抗原，成为抗生素治疗的替代方案(Rukavina,T et al.Infection and immunity vol.65,5(1997):1754-60.；Hsieh,Pei-Fang et al.Frontiers in microbiology vol.5 608.19 Nov.2014；Follador,Rainer et al.Microbial genomics vol.2,8e000073.25 Aug.2016；Szijártó,Valéria et al.International journal of medical microbiology:IJMM vol.306,2(2016):89-98.)。在克雷伯氏菌中共鉴定出9种不同的O抗原，其中O1抗原最为常见(Podschun,R,and U Ullmann.Clinical microbiology reviews vol.11,4(1998):589-603.；Hansen,D S et al.Journal of clinical microbiology vol.37,1(1999):56-62.)。
已有文献报道，针对肺炎克雷伯菌O1抗原的多克隆抗体和鼠单克隆IgG2a抗体在肺炎克雷伯菌感染的鼠模型中起到保护作用(Trautmann,M et al.Clinical and diagnostic laboratory immunology vol.4,5(1997):550-5.；Rukavina,T et al.Infection and immunity vol.65,5(1997):1754-60.)。现有技术(例如国际申请公开文本WO2017064258A1、WO2018075375A1)亦表明针对肺炎克雷伯菌O1抗原的抗体不仅可以通过调理吞噬杀伤(OPK)和/或补体依赖性杀伤(SBA)杀伤或杀死细菌，也可以中和血液中的内毒素，减少肺炎克雷伯菌对机体造成的损伤。因此开发新的抗LPS抗体以补充抗生素疗法至关重要。
本文提及的所有出版物、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文中。
申请概述
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:39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:46所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:40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:47所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:41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:48所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:42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:49所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC- CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:43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:50所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:44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:51所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％序列同一性。
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O1抗原的抗体，其包含：V H，其包含如氨基酸序列SEQ ID NO:45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:52所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
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O1抗原的抗体，其包含：V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38，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，如上所述任一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，其包含：V H，其包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％序列同一性。
在一些实施例中，分离的特异性结合肺炎克雷伯菌O1抗原的抗体与肺炎克雷伯菌O1抗原结合的Kd值为1pM至5nM。
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O1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O1抗原的抗体竞争性地结合肺炎克雷伯菌。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O1抗原的抗体，其与上述任一种分离的结合肺炎克雷伯菌O1抗原的抗体特异性地结合相同的表位。
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O1抗原的抗体，所述分离的特异性结合肺炎克雷伯菌O1抗原的抗体包含Fc片段。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O1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O1抗原的抗体是全长的IgG1、IgG2、IgG3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O1抗原的抗体是嵌合的、全人的或人源化的。在一些实施例中，所述分离的特异性结合肺炎克雷伯菌O1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、单链抗体(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，提供一种分离的特异性结合肺炎克雷伯菌O1抗原的抗体，所述抗体除结合肺炎克雷伯菌O1抗原外，还结合产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌O1抗原。
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O1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O1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O1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O1抗原的抗体的条件下培养上述任一种宿主细胞；和b)从所述宿主细胞中获得所表达的特异性结合肺炎克雷伯菌O1抗原的抗体。
在一些实施例中，提供一种治疗、预防或改善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O1抗原的抗体。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O1抗原的抗体在制备用于治疗、预防或改善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O1抗原的抗体或包含特异性结合肺炎克雷伯菌O1抗原的抗体的药物组合物在制备用于治疗、预防或改善疾病或病症的药物中的用途。在一些实施例中，所述疾病或病症与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植有关，包括院内感染、机会性感染、器官移植后感染以及与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染/定植相关的其他疾病或病症。在一些实施例中，所述疾病或病症选自例如，肺炎、***、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(PLA)、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。
同时还提供包含如上所述的任一种特异性结合肺炎克雷伯菌O1抗原的抗体的药物组合物、试剂盒以及生产制品。
附图说明
图1A-1D所示结果为肺炎克雷伯菌O1、O2血清型LPS结构示意图。图1A所示结果为肺炎克雷伯菌O1血清型KP19173菌株LPS结构示意图；图1B所示结果为肺炎克雷伯菌O1血清型KP19213菌株LPS结构示意图；图1C所示结果为肺炎克雷伯菌O2血清型KP19180菌株LPS结构示意图；
图1D所示结果为肺炎克雷伯菌O2血清型KP19203菌株LPS结构示意图。
图2A-2B所示结果为抗O1抗体G2或G7不与肺炎克雷伯菌O2血清型产生交叉反应。图2A所示结果为抗O1抗体G2或G7不与肺炎克雷伯菌O2血清型KP19180菌株LPS结合。图2B所示结果为抗O1抗体G2或G7不与肺炎克雷伯菌O2血清型KP19203菌株LPS结合。
图3所示结果为抗O1抗体G2或G7介导的调理吞噬杀伤活性。
图4A-4B所示结果为抗O1抗体G2或G7介导的血清杀菌活性。图4A为抗O1抗体G2介导的血清杀菌活性，图4B为抗O1抗体G7介导的血清杀菌活性。
图5A-5D所示结果为抗O1抗体G2或G7与其他肺炎克雷伯菌O1血清型LPS的结合。图5A所示结果为抗O1抗体G2或G7与临床上分离的肺炎克雷伯菌O1血清型KP1953菌株LPS的结合。图 5B所示结果为抗O1抗体G2或G7与临床上分离的肺炎克雷伯菌O1血清型KP1961菌株LPS的结合。图5C所示结果为抗O1抗体G2或G7与临床上分离的肺炎克雷伯菌O1血清型KP1962菌株LPS的结合。图5D所示结果为抗O1抗体G2或G7与临床上分离的肺炎克雷伯菌O1血清型KP1963菌株LPS的结合。
图6所示结果为抗O1抗体G2或G7在小鼠菌血症模型中的预防、保护作用。
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O1抗原的抗体分子(即抗O1抗体)。通过天然scFv酵母库筛选、适当设计的生物化学及生物学实验的组合，已经鉴定出能够结合肺炎克雷伯菌O1抗原并中和肺炎克雷伯菌内毒素(LPS)的高效抗体分子。本文给出的结果表明，与已知结合肺炎克雷伯菌O抗原的抗体MPG196、KPN70或G3-78相比，本申请中的抗体特异性结合肺炎克雷伯菌O1血清型，并且令人惊讶地是，在各种生物学实验中证明了本申请中的抗体部分性能方面甚至比同样特异性结合肺炎克雷伯菌O1抗原的MPG196更优。
本申请所提供的抗O1抗体包括，例如，全长抗O1抗体、抗肺炎克雷伯菌O1抗原单链抗体(scFvs)、抗O1Fc融合蛋白、多特异性(如双特异性)抗O1抗体、抗O1抗体免疫偶联物以及诸如此类的。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列DAWIS(SEQ ID NO:1)；HC-CDR2，其包含序列RIRSKTDGETREYAAPVNG(SEQ ID NO:7)；和HC-CDR3，其包含序列DPQWGI(SEQ ID NO:13)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RASQSVSSRHLA(SEQ ID NO:18)；LC-CDR2，其包含序列GASSRAT(SEQ ID NO:25)；和LC-CDR3，其包含序列QQYGSTPLT(SEQ ID NO:32)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列NAWMY(SEQ ID NO:2)的；HC-CDR2，其包含序列RIRSYSDGGTTDYAALVEG(SEQ ID NO:8)；和HC-CDR3，其包含序列PSGDFYPAS(SEQ ID NO:14)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RSSQNLLHSNGYNYLD(SEQ ID NO:19)；LC-CDR2，其包含序列LSSNRAS(SEQ ID NO:26)；和LC-CDR3，其包含序列MQALQTPYT(SEQ ID NO:33)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列NFWMT(SEQ ID NO:3)；HC-CDR2，其包含序列NINQGGTEGYYVDSVKG(SEQ ID NO:9)；和HC-CDR3，其包含序列HHGWKYNSGWRTAFDI(SEQ ID NO:15)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RASQGISNSLV(SEQ ID NO:20)；LC-CDR2，其包含序列GASKLHP(SEQ ID NO:27)；和LC-CDR3，其包含序列QQSGRSPYT(SEQ ID NO:34)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列DAWMS(SEQ ID NO:4)；HC-CDR2，其包含序列RIRSKADGETIEYAAHVAG(SEQ ID NO:10)；和HC-CDR3，其包含序列DPQWGI(SEQ ID NO:13)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列KSSQSLLHSGGKTHFY(SEQ ID NO:21)；LC-CDR2，其包含序列EVSNRFS(SEQ ID NO:28)；和LC-CDR3，其包含序列MQGTHWPPT(SEQ ID NO:35)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列NFWMT(SEQ ID NO:3)；HC-CDR2，其包含序列NINQGGTEGYYVDSVKG(SEQ ID NO:9)；和HC-CDR3，其包含序列HHGWKYNSGWRTAFDI(SEQ ID NO:15)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RTSQSISTHLN(SEQ ID NO:22)；LC-CDR2，其包含序列GASTLQN(SEQ ID NO:29)；和LC-CDR3，其包含序列QQSYRIPYS(SEQ ID NO:36)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列NYWMT(SEQ ID NO:5)；HC-CDR2，其包含序列SINQGGSEQYYVDSLKG(SEQ ID NO:11)；和HC-CDR3，其包含序列ADWMSIDH(SEQ ID NO:16)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RSSQSLVNSDGNIYLS(SEQ ID NO:23)；LC-CDR2，其包含序列QVSNRDS(SEQ ID NO:30)；和LC-CDR3，其包含序列MQGTHWPWT(SEQ ID NO:37)。
另一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗体，该抗O1抗体包含：重链可变结构域(V H)，所述V H包含：重链互补决定区(HC-CDR)1，其包含序列GYWMS(SEQ ID NO:6)；HC-CDR2，其包含序列NIKQDGSEQYYVDSVKG(SEQ ID NO:12)；和HC-CDR3，其包含序列DRGIKMGSVWYPSFDL(SEQ ID NO:17)；以及轻链可变结构域(V L)，所述V L包含：轻链互补决定区(LC-CDR)1，其包含序列RASRSISNYLN(SEQ ID NO:24)；LC-CDR2，其包含序列AASTLQS(SEQ ID NO:31)；和LC-CDR3，其包含序列QQSYSAPRT(SEQ ID NO:38)。
同时还提供编码抗O1抗体的核酸，包含抗O1抗体的组合物，以及制备和使用抗O1抗体的方法。
定义
如本文所述，“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，预防或延迟疾病恶化)，预防或延迟疾病的扩散(例如，病原菌全身扩散)，预防或延迟疾病复发，延迟或减缓疾病进展，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 疾病病理结果的减少(例如，对细菌感染而言，组织样本中细菌载量、炎性细胞浸润)。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”指的是包括防止发病的防止措施或降低发病风险的预防措施。
术语“抗体”包括全长抗体及其抗原结合片段。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)。本文所披露的抗体或抗原结合片段的CDR边界可以通过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***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)或IgA2(α2重链)。
如本文所述，术语“抗原结合片段”包括抗体片段，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(nanobody)、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。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。抗原结合片段还包括包含上述抗体片段的融合蛋白。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。
如本文所述，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。表位可由糖类、肽结构、脂肪酸、无机物质或它们的衍生物、有机物质、生化物质或它们的任何组合组成。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的相同表位。
如本文所用，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O1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O1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”方法。
如本文所述，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，例如抗体与靶标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。
如本文所述，一种“分离的”抗O1抗体是指一种抗O1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。
如本文所述，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”是指(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。
如本文所用，术语“CDR”或“互补决定区”意指重链和轻链多肽的可变域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，均包括在本文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文中，以用于本申请和可能包含在本文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结构域和一个轻链可变结构域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，即使单个可变域(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)”是在V H和V L域之间采用短接头(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，这样就使得可变域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、兔或非人类哺乳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所有，至少一个，通常两个可变域，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)。
本文所鉴定的多肽和抗体序列的“氨基酸序列同一性百分比(％)”或“同源性”被定义：在认为保守性取代属于序列同一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一性百分比，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，为了本文的目的，氨基酸序列同一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 PCTCN2022114384-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)；以及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)以及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.中进行了综述。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 Fc区的“CH1结构域”通常从118位氨基酸延伸到215位氨基酸(EU编号***)。
“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同种型的铰链区与IgG1序列比对。
人IgG Fc区的“CH2结构域”通常从231位氨基酸延伸到340位氨基酸。CH2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CH2结构域之间***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CH2结构域稳定。Burton,Molec.Immunol.22:161-206(1985)。
“CH3”结构域包括在Fc区内从C末端残基延伸到CH2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IC 50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IC 50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核细胞(PBMC)和自然杀伤性细胞(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区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区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***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文中。另见Idusogie et al.J.Immunol.164:4178-4184(2000)。
“效力”是以产生给定强度的作用所需的量来表示的化合物药理学活性的量度。它是指实现确定的生物效应所需的化合物的量；所需剂量越小药物越有效。抗O1抗体的效力可以使用例如本文所述的OPK试验或SBA试验来确定。
“调理吞噬杀伤”或“OPK”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)死亡。OPK活性检测的方法是本领域已知的，参见，例如DiGiandomenico,A.,et al.,InfectImmun 72,7012-7021(2004)。
杀伤也可以使用“血清杀菌测定”或“SBA”来测量。“SBA”亦是指在补体存在的情况下，通过补体激活以及“膜攻击复合物”的形成来裂解靶细胞。用SBA试验来评估抗体杀伤靶细胞效力的方法是本领域已知的，参见，例如Kobayashi,Scott D et al.“Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils.”mBio vol.9,2e00297-18.13 Mar.2018。
术语“克雷伯菌感染”以如下方式理解:克雷伯氏菌是革兰氏阴性细菌，它是肠杆菌科成员。它是一种无处不在的细菌，其也可以定植于人类宿主，通常在肠道或上呼吸道。作为机会致病菌，在免疫***没有对其适当控制的情况下，它可以从这些位点侵入无菌***点。在这些位点的不受控制的细菌复制将会诱发炎症，而炎症在很大程度上是由从克雷伯菌释放的内毒素(LPS)分子介导的。在菌血症的情况下，内毒素分子可以引发感染性休克。
“克雷伯菌定植”是指主体在可检测到的位点处具有足够高浓度的克雷伯菌，但细菌没有引起任何体征或症状。定植可以持续很长一段时间，并且受到机体对生物体的免疫反应、来自其它生物体的竞争、以及有时使用抗微生物剂的影响而造成溶解(分解)。
术语“中和”或“中和作用”广义上指的是抑制病原体的任何分子发挥其生物活性，而不管实现中和的机制是什么，如抑制克雷伯菌(例如肺炎克雷伯菌)感染主体、或抑制病原体通过产生内毒素促进感染、或抑制内毒素。例如，可以通过抗体抑制宿主粘膜表面克雷伯菌(例如肺炎克雷伯菌)定殖、侵入无菌体内部位和诱发不良生物信号(最糟的情况是诱发脓毒性休克)来实现中和。
从严格意义上来说，中和指的是抑制特异性LPS与其同源受体(例如与Toll-样受体-4复合物)结合，从而抑制其生物活性。这种中和效力一般采用本领域已知的标准试验测定，例如采用体外或体内中和试验，例如LAL试验或TLR-4报告细胞系测定。在这些试验中，例如采用比色法测定内毒性生物活性的抑制情况。
抵抗或中和克雷伯菌(例如肺炎克雷伯菌)的抗体干扰病原体和病原反应从而能够限制或预防感染、和/或改善这种感染引起的疾病状况，或者抑制克雷伯菌发病(例如肺炎克雷伯菌)，尤其是抑制克雷伯菌(例如肺炎克雷伯菌)散播和复制到宿主的无菌体腔内/身体部位或抑制其在宿主的无菌体腔内/身体部位内的散播和复制。在这方面，中和抗体还可理解为“保护性抗体”，是指在主动或被动免疫中观察到的引起机体对致病原的免疫反应的抗体。本发明所描述的中和或保护性抗体可用于治疗用途，例如用于预防或治疗，即预防、改善、治疗或至少部分抑制病原体诱导的疾病症状、副作用或进展。保护性抗体能够在治疗应用后(即施用在已有感染上)，通过例如 诱导血清杀菌或调理吞噬活动杀灭或阻止活克雷伯菌(例如肺炎克雷伯菌)细胞的复制，或从无菌的体内位点清除全部菌细胞或其LPS分子。预防性施用的保护性抗体可通过上文提到的机制或其它机制阻止感染的发生(例如阻止肺炎克雷伯菌从非无菌位点扩散到无菌体腔)。
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一性。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。
本文所公开的抗O1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”是指本文所公开的抗O1抗体或其组合物施用给主体时足以引起有益的效果或期望的结果(包括临床结果)的量或活性。因此，有效量或同义词取决于其施用的背景。有效量旨在表示足以治疗、预防或抑制此类疾病或病症的抗体或其组合物的量。在疾病的情况下，本文所述的治疗有效量的抗体特别地用于治疗、调节、减弱、逆转或影响疾病或病症，所述疾病或病症受益于抑制克雷伯菌(例如肺炎克雷伯菌)发病机理，例如粘膜表面的粘附和定植、无菌***点内的不受控制的复制以及细菌产物对宿主细胞的毒性。在感染时，本文公开的抗O1抗体或组合物能够抑制克雷伯菌生长(例如肺炎克雷伯菌)和/或杀死克雷伯菌(例如肺炎克雷伯菌)，抗O1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。
如本文所用的，“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。
本文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。
本文中提及“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。
如本文所用的，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型癌症，意味着该方法通常用于治疗除X型癌症之外的其他类型的癌症。
除非上下文另有明确说明，本文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。
抗O1抗体
一方面，本申请提供特异性结合肺炎克雷伯菌O1抗原的抗O1抗体。所述抗O1抗体包括，但不限于，人源化抗体，嵌合抗体，小鼠抗体，人抗体，以及本文所述的包含重链和/或轻链CDRs的抗体分子。一方面，本申请提供与肺炎克雷伯菌O1血清型结合的分离的抗体。预期的抗O1抗体包括，例如，全长抗O1抗体(如全长IgG1或IgG4)，抗O1单链抗体，抗O1Fc融合蛋白，多特异性(如双特异性)抗O1抗体，抗O1抗体-免疫偶联物，以及诸如此类的。在一些实施例中，抗O1抗体是全长抗体(如全长IgG1或IgG4)或其抗原结合片段，其特异性结合肺炎克雷伯菌O1抗原。在一些实施例中，抗O1抗体是Fab、Fab’、F(ab)’ 2、Fab’-SH、单链抗体(scFv)、Fv片段、dAb、Fd、纳米抗体(nanobody)、双链抗体(diabody)或线性抗体。在一些实施例中，特异性结合肺炎克雷伯菌O1抗原的抗体是指抗体与肺炎克雷伯菌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，抗O1抗体除结合肺炎克雷伯菌O1抗原外，还结合产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌O1抗原。在一些实施例中，非靶标是指不是克雷伯菌属O1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)技术或生物层干涉技术(BLI)。
尽管本文广泛地讨论了包含人序列的抗O1抗体(例如，包含人CDR序列的人重链和轻链可变结构域)，但同时也考虑了非人抗O1抗体。在一些实施例中，非人抗O1抗体包括本文所述的抗O1抗体的人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文所述的一种或多种人CDR序列产生重链和/或轻链可变结构域的序列，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，小猿，猕猴)等。在一些实施例中，非人抗O1抗体包括将一种或多种本文所述的人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O1抗体。
在一些实施例中，本文所述抗O1抗体特异性识别肺炎克雷伯菌O1抗原。在一些实施例中，所述抗O1抗体对克雷伯菌(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、 解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)O1血清型是特异性的，并且不与其他菌属或非O1血清型克雷伯菌发生交叉反应。
在一些实施例中，如本文所述的任一抗O1抗体，所述抗O1抗体包括抗体重链恒定区和抗体轻链恒定区。在一些实施例中，所述抗O1抗体包括IgG1型重链恒定区。在一些实施例中，所述抗O1抗体包括IgG2型重链恒定区。在一些实施例中，所述抗O1抗体包括IgG3型重链恒定区。在一些实施例中，所述抗O1抗体包括IgG4型重链恒定区。在一些实施例中，所述重链恒定区包含(包括由……组成或基本上由……组成)氨基酸序列SEQ ID NO:53。在一些实施例中，所述重链恒定区包含(包括由……组成或基本上由……组成)氨基酸序列SEQ ID NO:54。在一些实施例中，所述抗O1抗体包含κ轻链恒定区。在一些实施例中，所述轻链恒定区包含(包括由……组成或基本上由……组成)氨基酸序列SEQ ID NO:55。在一些实施例中，所述抗O1抗体包含λ轻链恒定区。在一些实施例中，所述轻链恒定区包含(包括由……组成或基本上由……组成)氨基酸序列SEQ ID NO:56。在一些实施例中，所述抗O1抗体包括抗体重链可变结构域和抗体轻链可变结构域。
在一些实施例中，所述抗O1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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32，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，所述抗O1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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及V L，所述V L包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:39中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:46中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:39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:46所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)；以及V L，其包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、 98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:39，以及V L，所述V L包含氨基酸序列SEQ ID NO:46。
在一些实施例中，所述抗O1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:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33，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，所述抗O1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:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:40中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:47中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:40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:47所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:40，以及V L，所述V L包含氨基酸序列SEQ ID NO:47。
在一些实施例中，所述抗O1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:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34，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，所述抗O1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:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列 SEQ ID NO:15；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:41中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:48中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:41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:48所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至少约90％例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:41，以及V L，所述V L包含氨基酸序列SEQ ID NO:48。
在一些实施例中，所述抗O1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:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35，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，所述抗O1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:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:42中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:49中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:42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:49所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％(例如至少91％、92％、93％、94％、95％、 96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:42，以及V L，所述V L包含氨基酸序列SEQ ID NO:49。
在一些实施例中，所述抗O1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:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36，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，所述抗O1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:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:43中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:50中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:43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:50所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:43，以及V L，所述V L包含氨基酸序列SEQ ID NO:50。
在一些实施例中，所述抗O1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:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37，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，所述抗O1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:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:44中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:51中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:44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:51所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:44，以及V L，所述V L包含氨基酸序列SEQ ID NO:51。
在一些实施例中，所述抗O1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:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38，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，所述抗O1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:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17；以及V L，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38。
在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:45中的1个、2个或3个HC-CDRs。
在一些实施例中，所述抗O1抗体包含：V L，所述V L包含氨基酸序列SEQ ID NO:52中的1个、2个或3个LC-CDRs。
在一些实施例中，所述抗O1抗体包含：V H，其包含如氨基酸序列SEQ ID NO:45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:52所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
在一些实施例中，所述抗O1抗体包含：V H，其包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述抗O1抗体包含：V H，所述V H包含氨基酸序列SEQ ID NO:45，以及V L，所述V L包含氨基酸序列SEQ ID NO:52。
在一些实施例中，上述氨基酸取代限于本文表4中所示的“示例性取代”。在一些实施例中，氨基酸取代限于本文表4中所示的“优选取代”。
在一些实施例中，本申请提供与本文所述的任一种抗O1抗体竞争性结合肺炎克雷伯菌O1抗原的抗体。在一些实施例中，提供能够与本文所述的任一种抗O1抗体竞争性地结合肺炎克雷伯菌O1抗原的抗体。在一些实施例中，提供抗O1抗体，其与包含V H和V L的抗O1抗体分子结合相同的表位，其中所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列，以及所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列。在一些实施例中，提供抗O1抗体，其与包含V H和V L的抗O1抗体竞争性地结合肺炎克雷伯菌O1抗原，其中所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列，以及所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列。
在一些实施例中，可以利用竞争实验来鉴定与本文所述的抗O1抗体竞争性结合肺炎克雷伯菌O1抗原的单克隆抗体。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文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，与本文所述的抗O1抗体竞争的抗体是嵌合抗体、人源化抗体或全人抗体。
示例性抗O1抗体序列如表2、表3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。包含如本文所述抗体的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。
表2示例性抗O1抗体CDR序列
Figure PCTCN2022114384-appb-000002
Figure PCTCN2022114384-appb-000003
表3示例性序列
Figure PCTCN2022114384-appb-000004
Figure PCTCN2022114384-appb-000005
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 PCTCN2022114384-appb-000006
其中
Figure PCTCN2022114384-appb-000007
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.)。
LPS
LPS是一种主要的表面抗原，其由包含不同数量的寡糖重复单元(RU)的O特异性多糖(O-specific polysaccharide，O-PS)、核心寡糖和脂质A构建而成。O-PS结构定义了克雷伯氏菌属菌株的O-血清型。与大多数革兰氏阴性菌相比，克雷伯氏菌属O抗原的变异性目前仅有 9种主要的O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.2,8 e000073.25 Aug.2016；)。O1和O2菌株表达的LPS包含由半乳糖(galactans,gal)均聚物构成的O-PS。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(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(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.)。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仅由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，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.5 608.19 Nov.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gal-III。对萃取的LPS或分离的O-PS进行结构学分析，揭示了可以同时存在gal-II和gal-III。最终，gal-II可以与gal-I或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gal-I或gal-III均聚物组成(Stojkovic,Katarina et al.Frontiers in microbiology vol.8 684.25 Apr.2017)。
全长抗O1抗体
在一些实施例中，所述抗O1抗体是全长抗O1抗体。在一些实施例中，所述全长抗O1抗体是IgA、IgD、IgE、IgG或IgM。在一些实施例中，所述全长抗O1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，所述全长抗O1抗体包含λ轻链恒定区。在一些实施例中，所述全长抗O1抗体包含κ轻链恒定区。在一些实施例中，所述全长抗O1抗体是全长的人抗O1抗体。在一些实施例中，所述全长抗O1抗体包含小鼠免疫球蛋白Fc序列。在一些实施例中，所述全长抗O1抗体包含已经改变的或以其他方式改变的Fc序列，使得其具有改变或增强的抗体依赖的细胞介导的细胞毒作用(ADCC)、补体依赖的细胞毒作用(CDC)和抗体依赖性细胞吞噬作用(ADCP)的效应功能。
因此，例如，在一些实施例中，提供一种包含IgG1恒定区的全长抗O1抗体，所述抗O1抗体与肺炎克雷伯菌O1抗原特异性结合。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG2恒定区的全长抗O1抗体，所述抗O1抗体与肺炎克雷伯菌O1抗原特异性结合。在一些实施例中，所述IgG2是人IgG2。在一些实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG3恒定区的全长抗O1抗体，所述抗O1抗体与肺炎克雷伯菌O1抗原特异性结合。在一些实施例中，所述IgG3是人IgG3。在一些实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG4恒定区的全长抗O1抗体，所述抗O1抗体与肺炎克雷伯菌O1抗原特异性结合。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列或其变体，所述变体包含至多约3个至多约3个(例如1、2或3个)氨基酸的取代；以及b)轻链可变 结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG2恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代。在一些实施例中，所述IgG2是人IgG2。在一些实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG3恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代，HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代。在一些实施例中，所述IgG3是人IgG3。在一些 实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代，和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列或其变体，所述变体包含至多约3个(例如1、2或3个)氨基酸的取代。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列，HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列，和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列，或者所述重链可变结构域的变体，其HC-CDR序列中包含至多约5个(例如1、2、3、4或5个)氨基酸的取代；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列，和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列，或者所述轻链可变结构域的变体，其LC-CDR序列中包含至多约5个(例如1、2、3、4或5个)氨基酸的取代。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基 酸序列，HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列，和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列，或者所述重链可变结构域的变体，其HC-CDR序列中包含至多约5个(例如1、2、3、4或5个)氨基酸的取代；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列，和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列，或者所述轻链可变结构域的变体，其LC-CDR序列中包含至多约5个(例如1、2、3、4或5个)氨基酸的取代。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列，HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列，和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列，和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含SEQ ID NOs:1-6中任一所示的氨基酸序列，HC-CDR2，其包含SEQ ID NOs:7-12中任一所示的氨基酸序列，和HC-CDR3，其包含SEQ ID NOs:13-17中任一所示的氨基酸序列；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含SEQ ID NOs:18-24中任一所示的氨基酸序列，LC-CDR2，其包含SEQ ID NOs:25-31中任一所示的氨基酸序列，和LC-CDR3，其包含SEQ ID NOs:32-38中任一所示的氨基酸序列。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及b)轻链可变域，所述轻链可变域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中所述抗O1抗体包含：a)重链可变结构域，所述重链可变结构域包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17；以及b)轻链可变结构域，所述轻链可变结构域包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域(V H)，所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:39-45中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及轻链可变结构域(V L)，所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:46-52中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG2恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域(V H)，所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:39-45中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及轻链可变结构域(V L)，所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:46-52中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述IgG2是人IgG2。在一些实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG3恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域(V H)，所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:39-45中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及轻链可变结构域(V L)，所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:46-52中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。在一些实施例中，所述IgG3是人IgG3。在一些实施例中，轻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:56组成。
在一些实施例中，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域(V H)，所述V H包含SEQ ID NOs:39-45中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:39-45中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性；以及轻链可变结构域(V L)，所述V L包含SEQ ID NOs:46-52中任一所示的氨基酸序列或其变体，所述变体与SEQ ID NOs:46-52中任一所示的氨基酸序列具有至少约90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域，其包含SEQ ID NOs:39-45中任一所示的氨基酸序列，以及轻链可变结构域，其包含 SEQ ID NOs:46-52中任一所示的氨基酸序列。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：重链可变结构域，其包含SEQ ID NOs:39-45中任一所示的氨基酸序列，以及轻链可变结构域，其包含SEQ ID NOs:46-52中任一所示的氨基酸序列。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至 少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG1恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％序列同一性。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:53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: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:56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:53组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:53组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％序列同一性。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:54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: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:56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:54组成以及 轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻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，提供一种包含IgG4恒定区的全长抗O1抗体，其中抗O1抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％序列同一性。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:54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: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:56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:54组成以及轻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，重链恒定区包含或由氨基酸序列SEQ ID NO:54组成以及轻链恒定区包含或由氨基酸序列SEQ ID NO:56组成。
结合亲和力
结合亲和力采用Kd、Koff、Kon或Ka表示。如本文所用，术语Koff是指抗体从抗原/抗体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/抗体复合物的结合速率常数。本文所用的平衡解离常数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位点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。抗体与细胞壁连接的情况，例如在酵母表达***中，相应的平衡解离速率常数采用EC 50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。
解离常数(Kd)可以作为反应抗体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，所述抗O1抗体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，抗O1抗体与肺炎克雷伯菌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，抗O1抗体与肺炎克雷伯菌O1抗原之间结合的Kd值为10 -7M至10 -13M。
在一些实施例中，抗O1抗体与非靶标之间结合的Kd值高于抗O1抗体与靶标的Kd值，并且本文中引用的一些实施例中，抗O1抗体与靶标(例如，肺炎克雷伯菌O1抗原)的结合亲和力高于抗O1抗体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抗原的抗原。在一些实施例中，抗O1抗体(针对肺炎克雷伯菌O1抗原)与非肺炎克雷伯菌O1抗原靶标结合 的Kd值间至少相差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，所述抗O1抗体与非靶标结合的Kd值为10 -1M至10 -6M(例如10 -1M至10 -6M、10 -1M至10 -5M、10 -2M至10 -4M)。在一些实施例中，所述非靶标是指非肺炎克雷伯菌O1抗原的抗原。因此，在一些实施例中，抗O1抗体与非肺炎克雷伯菌O1抗原靶标之间结合的Kd值为10 -1M至10 -6M、1×10 -1M至5×10 -6M、10 -1M至10 -5M、1×10 -1M至5×10 -5M、10 -1M至10 -4M、1×10 -1M至5×10 -4M、10 -1M至10 -3M、1×10 -1M至5×10 -3M、10 -1M至10 -2M、10 -2M至10 -6M、1×10 -2M至5×10 -6M、10 -2M至10 -5M、1×10 -2M至5×10 -5M、10 -2M至10 -4M、1×10 -2M至5×10 -4M、10 -2M至10 -3M、10 -3M至10 -6M、1×10 -3M至5×10 -6M、10 -3M至10 -5M、1×10 -3M至5×10 -5M、10 -3M至10 -4M、10 -4M至10 -6M、1×10 -4M至5×10 -6M、10 -4M至10 -5M、10 -5M至10 -6M。
在一些实施例中，当提及抗O1抗体以高结合亲和力特异性地识别肺炎克雷伯菌O1抗原靶标，并以低结合亲和力结合非靶标时，所述抗O1抗体与肺炎克雷伯菌O1抗原靶标结合的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、10 -10M至10 -12M)，并且与非靶标结合的Kd值为10 -1M至10 -6M(例如10 -1M至10 -6M、10 -1M至10 -5M、10 -2M至10 -4M)。
在一些实施例中，当提及抗O1抗体特异性地识别肺炎克雷伯菌O1抗原时，将所述抗O1抗体的结合亲和力与对照抗O1抗体(例如MPG196)或其他抗O抗原抗体(例如KPN70或G3-78)的结合亲和力进行比较。在一些实施例中，对照抗O1抗体或抗O抗原抗体与肺炎克雷伯菌O1抗原之间结合的Kd值可以是本申请所述的抗O1抗体与肺炎克雷伯菌O1抗原之间结合的Kd值的至少2倍，例如2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、10-100倍、100-1000倍、10 3-10 4倍。
核酸
编码抗O1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)编码全长抗O1抗体的核酸，包括本文所述的任一种全长抗O1抗体。在一些实施例中，本文所述的抗O1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)。
同时本文还考虑了包含抗O1抗体的分离的宿主细胞，编码抗O1抗体多肽组分的分离的核酸，或者包含编码本文所述的抗O1抗体多肽组分的核酸的载体。
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O1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***到其中的载体。
简言之，将编码抗O1抗体的天然或合成的核酸***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O1抗体(例如全长的抗O1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文。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***，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***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***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***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***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，抗O1抗体的表达是可诱导的。在一些实施例中，编码抗O1抗体的核酸序列可操作的连接到诱导型启动子上，包括本文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***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O1抗体的诱导型启动子***为Tet***。在一些实施例中，用于表达抗O1抗体的诱导型启动子***为大肠杆菌lac抑制***。
本申请所采用的一个示例性诱导型启动子***为Tet***。该***是基于Gossen等(1993)描述的Tet***。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***的其它具体变体，包括以下的“Tet-Off”和“Tet-On”***。在Tet-off***中，转录在Tc或Dox存在下是失活的。在该***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***中，转录在Tc或Dox存在下是激活的。Tet-On***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转录激活结构域组成的融合蛋白。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***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***是大肠杆菌的lac阻遏物***(参见Brown et al.,Cell 49:603-612(1987))。Lac阻遏物***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(参见,Ui-Tel et al.,2000 FEBS Letters 479:79-82)。合适的表达***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，把可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，提供编码本文所述的任一种全长抗O1抗体的核酸。在一些实施例中，所述核酸包括编码全长抗O1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***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***，例如高分子复合物、纳米胶囊、微球、磁珠和以脂质为基础的***，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***是脂质体(例如，人工膜囊)。
在使用非病毒递送***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存在或不存在，例如通过免疫学方法(ELISAs和Western blots)或者通过本文所述的实验来进行鉴定均落入本申请范围内。
抗O1抗体的制备
在一些实施例中，所述抗O1抗体是单克隆抗体或源于单克隆抗体。在一些实施例中，所述抗O1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，所述抗O1抗体进一步包括来自单克隆抗体的CH1和CL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、噬菌体展示方法或应用重组DNA法。此外，示例性的噬菌体展示法在本文及以下的实施例中进行了描述。
在杂交瘤细胞法中，通常采用免疫剂免疫仓鼠、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***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。通常采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，根据本文所述的任一抗O1抗体，所述抗O1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肺炎克雷伯菌O1抗原的抗O1抗体部分的方法来制备所述的抗O1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。
与靶标肺炎克雷伯菌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肺炎克雷伯菌O1抗原的迭代结合进行筛选，所述靶标肺炎克雷伯菌O1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肺炎克雷伯菌O1抗原的噬菌体克隆。富集的噬菌体克隆与靶标肺炎克雷伯菌O1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。
筛选抗体文库的另一种方法是在酵母细胞表面展示蛋白质。Wittrup等(美国专利6,699,658和6,696,251)开发了一种酵母细胞展示文库的方法。在此酵母展示***中，一个组分包括锚定在酵母细胞壁上的酵母凝集素蛋白(Aga1)，另一个组分包括凝集素蛋白Aga2的第二个亚基，该亚基可以通过二硫键与Aga1蛋白结合进而展示在酵母细胞表面上。通过将Aga1基因整合到酵母染色体中来表达Aga1蛋白。将单链可变片段(scFv)文库与酵母展示质粒中的Aga2基因融合，将其转化后，该文库由于附加的营养标记的存在可保留在酵母中。Aga1和Aga2蛋白均在半乳糖诱导型启动子的控制下表达。
人抗体V基因库(V H和V K片段)是使用一组简并引物通过PCR方法获得(Sblattero，D.and Bradbury,A.Immunotechnology 3,271-278 1998)。PCR模板来自可商购的RNA或cDNA，包括PBMC，脾脏，***，骨髓和扁桃体。将独立的V H和V K PCR文库合并后，通过重叠延伸PCR将其组装成scFv形式(Sheets，M.D.et al,Proc.Natl.Acad.Sci.USA 95,6157–6162 1998)。为了构建酵母scFv展示文库，通过同源重组将所得的scFv PCR产物克隆到酵母中的酵母展示质粒中。(Chao,G,et al,Nat Protoc.2006；1(2):755-68.Miller KD,et al.Current Protocols in Cytometry 4.7.1-4.7.30,2008)。
可以利用哺乳动物细胞展示***来筛选抗O1抗体，其中抗体部分展示在细胞表面上并通过抗原导向的筛选方法分离出特异性靶向肺炎克雷伯菌O1抗原的抗体(如U.S.patent No.7,732,195B2中所述)。可以建立展示大量人类IgG抗体基因的中国仓鼠卵巢(CHO)细胞文库，并将其用于发现表达高亲和力抗体基因的克隆。已开发出另一种展示***，该***通过可变剪接使同一蛋白同时在细胞表面展示和分泌，其中展示的蛋白表型保持与基因型相关，使得可同时在生物物理和基于细胞功能的分析中表征该分泌的可溶性抗体。该方法克服了先前哺乳动物细胞展示的许多局限性，能够直接筛选和成熟化全长的、糖基化的IgGs形式的抗体(Peter M.Bowers,et al,Methods2014,65：44-56)。瞬时表达***适用于在抗体基因恢复之前进行的单轮抗原选择，因此对于从较小文库中选择抗体最有用。稳定的外显体载体提供了一种有吸引力的选择。外显体载体可以高效转染并稳定地维持在低拷贝数，从而允许多轮淘选以及更复杂抗体库的解析。
IgG文库是基于分离自一群人类供体的种系序列V基因片段与重排的(D)J区域的连接构建而成。将从人体血液样本中收集的RNA反转录为cDNA，使用V H和V K特异性引物扩增V H和V K片段，并通过凝胶提取纯化。将V H和V K片段分别亚克隆到包含IgG1或K恒定区的展示载体中，然后电穿孔或转导293T到细胞，从而制备IgG文库。为了制备scFv抗体展示文库，连接V H和V K以产生scFv，然后亚克隆到展示载体中，再将其电穿孔或转导293T细胞。众所周知，IgG文库是基于分离自一群供体的种系序列V基因片段与重排的(D)J区域构建而成，供体可以是小鼠，大鼠，兔或猴。
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肺炎克雷伯菌O1抗原特异性酵母克隆可以作为这种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，例如用编码序列取代人重链和轻链恒定区和/或用框架区替换同源非人序列(U.S.Patent No.4,816,567；Morrison et al.,supra)，或通过共价键连接免疫球蛋白的编码序列的全部或部分非免疫球蛋白多肽的编码序列。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域中的一个抗原结合位点，形成嵌合的二价抗体。
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。
具有所需结合特异性(抗体-抗原结合位点)的抗体可变域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，CH2和CH3区。在一些实施例中，包含轻链结合必要位点的第一重链恒定区(CH1)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***进独立的表达载体中，并共转染至合适的宿主生物中。
全人和人源化抗体
所述抗O1抗体(如全长的抗O1抗体)可以是全人抗体或人源化抗体。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，通常两个可变域，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”可变域。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，其可变域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。
全人抗体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人源抗体，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。
全人抗体也以通过体外活化B细胞(见U.S.Patents 5,567,610 and 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O1抗体变体
在一些实施例中，本文提供的抗O1抗体变体(例如，全长的抗O1抗体)的氨基酸序列也在考虑中。例如，可能需要改善抗体的结合亲和力和/或其它生物学活性。抗体变体的氨基酸序列可以通过在编码抗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，抗体氨基酸序列中残基的缺失和/或***和/或取代。可以通过氨基酸残基缺失、***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O1抗体变体。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，或改善的ADCC或CDC。
保守取代如下表4所示。
表4保守取代
原始残基 示例性取代 优选取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp,Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；Norleucine Leu
Leu(L) Norleucine；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；Norleucine Leu
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、含有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，基于中和活性或结合亲和力)的变体。可以在HVRs区进行改变(例如，取代)来获得改善的中和活性或结合亲和力。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、***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，本文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。
一种有用的可以鉴定出抗体中能被靶向性突变的氨基酸残基或区域的方法称为“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***，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***1个或多个氨基酸残基。末端***的例子包括N末端具有甲硫氨酰残基的抗体。抗体分子的其它***变体，包括在抗体分子N-末端或C-末端融合一个酶(例如，ADEPT)或增加抗体血清半衰期的多肽。
Fc区变体
在一些实施例中，将一个或多个氨基酸修饰引入本文所述的抗体(例如，全长抗O1抗体或抗O1抗体融合蛋白)的Fc区，从而产生Fc区变体。在一些实施例中，Fc区变体具有增强的ADCC、CDC和/或ADCP效能，通常与结合Fc的受体(FcRs)有关。在一些实施例中，Fc区变体具有降低的ADCC、CDC和/或ADCP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文。
抗体可以在Fc区中被修饰和/或改变以便提供所希望的效应子功能或血清半衰期。如在下面几个部分中更详细地讨论的，在具有适当的Fc区的情况下，在细胞表面上结合的裸抗体可以通过如下方式诱导细胞毒性:经由抗体依赖性细胞毒性(ADCC)；通过在补体依赖性细胞毒性(CDC)中募集补体；或通过募集表达一个或多个效应子配体的非特异性细胞毒性细胞，这些效应子配体识别细菌(例如肺炎克雷伯菌)上结合的抗体并且随后在抗体依赖性细胞介导的吞噬作用(ADCP)中引起细胞的吞噬作用；或通过其他一些机制。在期望消除或减少效应子功能(例如减少副作用或治疗并发症)的情况下，可以使用经修饰和/或改变的Fc区，例如增加对FcRn的结合亲和力并增加血清半衰期。Fc区也可以与诸如PEG或白蛋白等部分轭合以增加血清半衰期。
抗体依赖的细胞介导的细胞毒作用(ADCC)是治疗性抗体针对病变细胞的作用机制。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体(例如，抗O1抗体)结合，免疫***的效应细胞主动裂解靶细胞(例如，感染细胞)。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，释放细胞裂解颗粒，随后靶细胞凋亡。
在一些实施例中，本申请还提供抗O1抗体变体(例如全长抗O1抗体变体)，其包含具有部分但不是全部的效应功能Fc区，使得其在体内具有延长的半衰期，然而特定的效应功能(例如 CDC或ADCC)是非必需的或有害的，这种抗O1抗体成为本申请理想的候选。通过在体外和/或体内进行细胞毒性检测来确认CDC和/或ADCC活性的减少/消除。例如，通过Fc受体(FcR)结合试验来确认抗体缺乏FcγR结合能力(因此可能缺乏ADCC活性)但依然保留FcRn的结合能力。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WO 2006/029879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；WO 2004/056312，和Shields et al.,J.Biol.Chem.9(2):6591-6604(2001))。
在一些实施例中，提供一种抗O1抗体(例如全长的抗O1抗体)变体，其包含具有一个或多个能够增强ADCC效应的氨基酸取代的Fc区变体。在一些实施例中，Fc区变体包含一个或多个能够增强ADCC效应的氨基取代，这些取代的位置在Fc区的298、333和/或334位(EU残基编号)。在一些实施例中，所述抗O1抗体(例如，全长的抗O1抗体)变体包括在Fc区的S298A，E333A和K334A位氨基酸取代。
在一些实施例中，Fc区的改变导致C1q结合和/或补体依赖性细胞毒作用(CDC)的改变(即增强或减弱)，参见U.S.Pat.No.6,194,551,WO 99/51642，和Idusogie et al.,J.Immunol.164:4178-4184(2000)中所述。
在一些实施例中，提供一种抗O1抗体(例如全长的抗O1抗体)变体，其包含具有一个或多个氨基酸取代的Fc区变体，能够延长半衰期或增强与Fc受体(FcRn)的结合。具有延长半衰期和改善FcRn结合的抗体在US 2005/0014934A1(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区变体的例子。
本申请考虑了包括本文所述的任一种Fc变体或其组合的抗O1抗体(例如全长抗O1抗体)。
糖基化变体
在一些实施例中，对本文所提供的抗O1抗体(例如全长抗O1抗体)进行改变，以增加或降低抗NGF抗体糖基化的程度。通过改变抗NGF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抗O1抗体上的糖基化位点。
其中抗O1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CH2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，可对本申请的抗O1抗体进行寡糖修饰，从而产生具有某些改进特性的抗O1抗体变体。
与Fc区的CH2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FcRIIIa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CH2结构域是非糖基化的。在一些实施例中，通过对CH2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，提供抗O1抗体(例如全长的抗O1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，本文提供抗O1抗体，其相对于野生型CHO细胞产生的相同抗O1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，所述抗O1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，该抗O1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，所述抗O1抗体的N-连接聚糖不 包含岩藻糖，即，其中抗O1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；WO 2005/053742；WO 2002/031140；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 2004/056312 A1,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)。
抗O1抗体(例如全长抗O1抗体)变体进一步提供二等分寡糖，例如，其中连接于抗O1抗体Fc区的双触角寡糖被GlcNAc等分。这种抗O1抗体(例如全长的抗O1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。还提供抗O1抗体(例如全长的抗O1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。这类抗O1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，所述抗O1抗体(例如全长抗O1抗体)变体包含能与FcγRIII结合的Fc区。在一些实施例中，包含Fc区的所述抗O1抗体(例如全长抗O1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区的其他相同抗O1抗体(例如全长抗O1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抗O1抗体(例如全长抗O1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，取代残基出现在抗O1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抗O1抗体的可及位点，可以用于将该抗O1抗体与其它部分偶联，例如药物部分或接头-药物部分，来制备如本文中进一步描述 的抗肺炎克雷伯菌O1抗原免疫偶联物。半胱氨酸工程化的抗O1抗体(例如，全长抗O1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，本文所提供的抗O1抗体(例如全长抗O1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。适用于衍生化抗O1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。连接在抗O1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，需要改进抗O1抗体的特性或功能，抗O1抗体衍生物是否用于特定条件下的治疗等。
药物组合物
本文还提供包含任一种抗O1抗体(例如全长抗O1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文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，包含本文所述的任一种抗O1抗体和药学上可接受的载体。
可通过混合具有所需纯度的抗O1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))获得合适的抗O1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文。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文中待治疗个体。阳离子脂质体或脂质体可以用于将本申请中的抗O1抗体递送至细胞。
本文所述的制剂除包含抗O1抗体(例如全长抗O1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，除了抗O1抗体之外，可能需要进一步包含抗生素、类固醇和非类固醇炎症抑制剂以及其他抗菌剂、抗炎剂。这些分子以对预期目的有效的量组合存在。其它物质的有效量取决于制剂中的抗O1抗体的含量，疾病或病症或治疗的类型，以及如上所述的其它因素。这些药物通常以与本文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。
所述抗O1抗体(例如，全长抗O1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***(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。
可以制备抗O1抗体(例如，全长抗O1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 DEPOTTM(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抗O1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，所述抗O1抗体(例如全长抗O1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。
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。
使用抗O1抗体的治疗或预防方法
抗O1抗体(例如，全长的抗O1抗体)和/或本申请所述的组合物可以施用于个体(例如，哺乳动物，如人类)来治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病和/或病症(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾 病)，这些疾病包括但不限于肺炎、***、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(PLA)、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。因此，本申请在一些实施例中，提供一种治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病和/或病症(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾病)的方法，包括向个体施用有效量的包含抗O1抗体(例如，全长的抗O1抗体)的组合物(例如，药物组合物)，例如本文所述的任一种抗O1抗体(例如，全长的抗O1抗体)。在一些实施例中，所述个体是人类。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含特异性结合肺炎克雷伯菌O1抗原的抗O1抗体(例如，全长抗O1抗体)的药物组合物。在一些实施例中，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，肺炎、***、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(PLA)、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，所述个体是人类。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克拉雷伯O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗O1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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。 在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、 臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:5，HC-CDR2，其包含氨基酸序列SEQ ID  NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
例如，在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体(例如，全长抗O1抗体)的药物组合物，其中所述抗体包含：V H，所述V H包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38，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，所述抗O1抗体是全长抗体。在一些实施例中，所述全长抗O1抗体是IgG1或IgG4抗体。在一些实施例中，所述疾病或病症选自例如，例如，肺炎、***、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(PLA)、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，所述个体是人类。
在一些实施例中，提供一种用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或定植相关的疾病(例如，院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的个体的方法，包括向所述个体施用有效量的包含抗O1抗体的组合物，其中所述抗体包括：V H，所述V H包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％序列同一性；以及V L，所述V L包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％序列同一性。
在一些实施例中，本文所述抗O1抗体是包含IgG1或IgG4恒定区的全长抗O1抗体。在一些实施例中，所述IgG1是人IgG1。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:53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:54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: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:56组成。
在一些实施例中，本文所述任一种治疗或预防的方法，所述方法进一步提供与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关疾病和/或病症的治疗性或预防性的效果。在一些实施例中，所述方法可预防克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染。
在一些实施例中，所述个体是哺乳动物(例如人、非人灵长类、大鼠、小鼠、牛、马、猪、绵羊、山羊、狗、猫等)。在一些实施例中，所述个体是人类。在一些实施例中，所述个体是临床患者、临床试验志愿者、实验动物等。在一些实施例中，所述个体年龄小于60岁(包括例如小于50、40、30、25、20、15或10岁)。在一些实施例中，所述个体年龄大于60岁(包括例如大于70、80、90或100岁)。在一些实施例中，所述个体是被诊断为克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染或免疫受损、免疫抑制以致易受克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的主体或其接触人。
在一些实施例中，所述个体存在与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷 伯菌和/或鼻硬结克雷伯菌)感染相关的一个或多个危险因素。例如，在一些实施例中，所述个体已暴露或破坏皮肤粘液层。在一些实施例中，所述个体有一种或多种烧伤。在一些实施例中，所述个体有一种或多种手术伤口。在一些实施例中，所述个体有皮肤疾病。在一些实施例中，所述个体有异物植入，例如，但不限于一个呼吸器或导管。在一些实施例中，所述个体被诊断患有或在遗传上容易患上免疫缺陷疾病，包括但不限于HIV感染、AIDS和/或中性粒细胞缺乏症。在一些实施例中，所述个体接受过一种或多种形式的化疗。在一些实施例中，所述个体接受过一种或多种形式的肾上腺糖皮质激素治疗。在一些实施例中，所述个体接受过一种或多种形式的化疗。在一些实施例中，所述个体被诊断患有或在遗传上容易患上癌症、糖尿病和/或慢性结构性肺病(例如囊性纤维化或COPD)。在一些实施例中，所述个体被诊断患有或在遗传上容易患上消化***和/或其他器官的菌群失调。在一些实施例中，所述个体有与上述一种或多种疾病或病症相关的一种或多种危险因素。
院内感染、机会性感染、器官移植后感染以及与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾病的许多诊断方法和这些疾病的临床描述在本领域是已知的。这类方法包括，但不限于，例如免疫组化、ELISA、PCR、蛋白免疫印迹以及荧光原位杂交(FISH)。
在一些实施例中，本申请所述抗O1抗体(例如全长抗O1抗体)和/或组合物与第二、第三或第四药剂(例如采用抗生素、类固醇和非类固醇炎症抑制剂，和/或其它抗菌剂或抗炎剂)联合使用来治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的疾病。
在一些实施例中，所述抗生素是一种青霉素、一种头孢菌素、一种碳青霉烯、一种氟喹诺酮、一种氨基糖苷、一种单胺菌、一种多粘菌素、一种含有β-内酰胺酶抑制剂的抗生素组合物，或它们的任意组合物。在一些实施例中，所述抗生素是头孢吡肟、头孢他啶、头孢匹罗、亚胺培南、美罗培南、替卡西林、哌拉西林、阿洛西林、卡苄西林、美洛西林、阿曲南、妥布霉素、庆大霉素、阿米卡星、环丙沙星、左氧氟沙星、头孢哌松舒巴坦、哌拉西林-他唑巴坦、磷霉素或其任何组合。在一些实施例中，所述抗生素是亚胺培南、妥布霉素、环丙沙星、美罗培南或阿曲南中的一种或多种。在一些实施例中，所述抗生素是庆大霉素、氨苄西林或卡那霉素中的一种或多种。
抗O1抗体的给药剂量和方法
施用于个体(例如人)的抗O1抗体(例如分离的抗O1抗体)组合物的剂量可能因特定组合物、给药方式和治疗疾病类型的不同而不同。在一些实施例中，组合物(例如，包含抗O1抗体的组合物)的量可在院内感染、机会性感染、器官移植后感染以及与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾病治疗中有效产生客观 响应(例如，部分响应或完全响应)。在一些实施例中，抗O1抗体组合物的量足以在个体中产生完全响应。在一些实施例中，抗O1抗体组合物的量足以在个体中产生部分响应。在一些实施例中，抗O1抗体组合物的给药剂量(例如当单独施用时)足以在使用抗O1抗体组合物治疗的个体群体中产生高于20％、25％、30％、35％、40％、45％、50％、55％、60％、64％、65％、70％、75％、80％、85％或90％的总响应率。个体对本文所述治疗方法的响应可通过，例如，利用革兰氏染色或其他表型测试方法对克雷伯菌的检测来确定。
在一些实施例中，组合物(例如包含分离的抗O1抗体的组合物)的量足以延长个体的无进展生存期。在一些实施例中，组合物的量足以延长个体的总体生存期。在一些实施例中，在使用抗O1抗体组合物治疗的个体群体中，组合物的量(例如当单独施用时)足以产生高于50％、60％、70％或77％的临床益处。
在一些实施例中，组合物(例如包含分离的抗O1抗体的组合物)的量，单独使用或与第二，第三、和/或第四药剂联合使用时，与同一受试者治疗前相比或与未接受治疗的其它受试者的相应器官负荷相比，其足以减轻克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌)器官负荷的强度至少为10％、20％、30％、40％、50％、60％、70％、80％、90％、95％或100％。可以采用标准方法来测量该疗效的大小，例如纯化酶的体外检测、基于细胞的检测、动物模型或人体试验。
在一些实施例中，当将组合物施用于个体时，组合物中抗O1抗体(例如全长的抗O1抗体)的量低于引起毒性效应(即，一种高于临床可接受毒性水平的效应)的水平，或者处于潜在副作用可以控制或耐受的水平。
在一些实施例中，遵循相同的给药方案，组合物的量接近的组合物的最大耐受剂量(MTD)。在一些实施例中，组合物的量高于MTD的80％、90％、95％或98％。
在一些实施例中，组合物中抗O1抗体(例如全长的抗O1抗体)的含量在0.001μg到1000μg的范围之内。
在如上所述任一个实施例中，组合物中的抗O1抗体(例如全长的抗O1抗体)的有效量，按照体重时计算，为0.1μg/kg到100mg/kg的范围之内。
抗O1抗体组合物可通过多种途径施用于个体(如人类)，包括，例如静脉注射、动脉内给药、腹腔注射、肺内给药、口服给药、吸入给药、血管内给药、肌肉注射、气管内给药、皮下注射、眼内给药、鞘内给药、粘膜给药或经皮给药。在一些实施例中，使用组合物的缓释制剂。在一些实施例中，组合物通过静脉给药。在一些实施例中，组合物通过动脉给药。在一些实施例中，组合物通过腹膜内给药。在一些实施例中，组合物通过肝内给药。在一些实施例中，组合物通过肝动脉输注给药。在一些实施例中，组合物施用于远离第一病灶的部位。
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，所述物质用于治疗或预防个体中克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、 土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染，或者用于递送抗O1抗体(例如一种全长抗O1抗体)到被表达LPS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或预防本文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O1抗体。标签或包装说明书标示了该组合物可以用于治疗或预防特定病症。标签或包装说明书进一步包含给患者施用抗O1抗体组合物的说明书。包括联合治疗的制品和试剂盒均在本文的考虑范围之内。
包装说明书是指通常包含在治疗或预防产品的商业包装内的说明书，其包含关于与这些治疗或预防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或预防与克雷伯菌，优选克雷伯菌O1血清型感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的其他疾病)。在一些实施例中，包装说明书标明该组合物可以用于治疗或预防以下的疾病，包括肺炎、***、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(PLA)、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。
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。
同时还涉及可用于各种目的的试剂盒，例如用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)，或者用于递送抗O1抗体(例如全长抗O1抗体)到被表达LPS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，其包含抗O1抗体组合物(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(例如本文所述的药剂)和/或与本文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或预防个体的描述。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，试剂盒包括一种包含抗O1抗体(例如全长的抗O1抗体)的组合物。在一些实施例中，试剂盒包括：a)包含本文所述的任一种抗O1抗体的组合物，和b)至少一种有效 量的其它药剂，其能够增强抗O1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)包含本文所述的任一种抗O1抗体的组合物，和b)向个体施用抗O1抗体组合物用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型(例如肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌)感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的使用说明书。在一些实施例中，试剂盒包括：a)包含本文所述的任一种抗O1抗体的组合物，和b)至少一种有效量的其它药剂，其能够增强抗O1抗体的效果(如治疗效果、检测效果)和c)向个体施用抗O1抗体组合物和其它物质用于治疗或预防与克雷伯菌，优选克雷伯菌O1血清型感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)的使用说明书。所述抗O1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抗O1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)编码抗O1抗体(例如全长的抗O1抗体)的核酸。在一些实施例中，试剂盒包含：a)一种(或一组)编码抗O1抗体(例如全长的抗O1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)编码抗O1抗体(例如全长的抗O1抗体)的核酸，和b)使用说明书，适用于：i)在宿主细胞中表达抗O1抗体，ii)制备包含抗O1抗体的组合物，和iii)向个体施用包含抗O1抗体的组合物来治疗或预防与克雷伯菌，优选克雷伯菌O1血清型感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)。在一些实施例中，试剂盒包括：a)一种(或一组)编码抗O1抗体(例如全长的抗O1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)在宿主细胞中表达抗O1抗体，ii)制备包含抗O1抗体的组合物，和iii)向个体施用包含抗O1抗体的组合物来治疗或预防与克雷伯菌，优选克雷伯菌O1血清型感染相关的疾病(例如院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染相关的其他疾病)。
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。因此，本申请还提供制品，其包括小瓶、瓶子、广口瓶、软包装(例如密封的聚酯薄膜或塑料袋)等。
关于抗O1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等。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文所述的抗O1抗体(例如全长的抗O1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抗O1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，医院药房和复方药房。
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-gal Ⅰ和D-gal Ⅱ两类重复单元的肺炎克雷伯菌O1血清型(图1A)；以KP19213代表LPS包含D-gal Ⅲ和D-galⅡ两类重复单元的肺炎克雷伯菌O1血清型(图1B)；以KP19180代表LPS包含D-gal Ⅰ重复单元的肺炎克雷伯菌O2血清型(图1C)；以KP19203代表LPS包含D-gal Ⅲ重复单元的肺炎克雷伯菌O2血清型(图1D)。在本发明的实施例中，以专利申请CN109689090A中披露的抗O2抗体KPN70(结合D-gal Ⅰ重复单元)、专利申请CN107371365A中披露的抗O抗原抗体G3-78(结合D-gal Ⅲ重复单元)、和/或专利申请WO2018/029356Al中披露的抗O1抗体MPG196(结合D-gal II重复单元)作为对照抗体。
实施例1：制备LPS、O-PS并筛选抗O1的单链抗体(scFv)
制备LPS
挑取肺炎克雷伯菌单克隆(例如KP19173、KP19213、KP19180或KP19203等)接种到2×YT培养基中进行扩大培养。12000rpm、4℃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次以破碎菌体。采用热酚水法提取LPS：首先将555mL 90％苯酚溶液加入到600mL的细菌悬液中，在65℃水浴条件下不断搅拌，作用1h后取出。在冷水浴中冷却到4℃后，20000g、4℃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。将混合物冰浴10min后，明显观察到有白色点状沉淀析出。20000g、4℃离心1h，弃去上清。将沉淀用70％乙醇完全重悬，13000rpm、4℃离心15min，弃去上清。将离心管置于超净台吹干后，即得LPS粗提物。将粗提物配置成2％(w/v)的溶液，加入等量的95％乙醇，边加边搅拌。12000rpm离心30min，去除沉淀(重复两次)。取上清置于新管中，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。冰浴10min，20000g、4℃离心30min，弃去上清。将沉淀用100mL 70％乙醇重悬，20000g、4℃离心30min，弃去上清。将沉淀抽真空干燥后备用。
制备O-PS
称取1g由上述过程中制备的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。沉淀为脂质A，上清为O-PS。取上清，采用抽真空离心的方法浓缩O-PS；随后用水溶解备用。
制备生物素化标记的O-PS
取适量的O-PS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O-PS，利用硫酸-苯酚比色法检测O-PS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O-PS质量比为1.35:1，称取适量的Amine-PEG11-Biotin加入O-PS溶液中，37℃孵育4天，分别在1h、24h、48h、72h时，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O-PS的溶剂更换为PBS，所得溶液即为生物素化标记的O-PS(命名为Bio-OPS)。
筛选抗O1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 K特异性引物扩增V H和V K片段，经过胶回收纯化后，连接V H和V K，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。
筛选抗O1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O-PS的scFvs。简言之，采用MACS磁珠分选，对结合肺炎克雷伯菌O-PS的酵母细胞进行富集。将1000OD的酵母细胞在2500g下离心5分钟，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40-48小时。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-OPS的PBSM溶液重悬细胞沉淀，4℃孵育1小时。经过离心和PBSM洗涤后，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30分钟。2500g离心5分钟，弃去上清，用5-10倍体积的PBSM溶液重悬沉淀。每次取7mL细胞悬液加入到柱子上，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筛选抗O1抗原的单链抗体(scFv)：对经MACS淘选后富集的酵母进行流式细胞术分选(FACS)。简言之，将SGCAA培养基中诱导的酵母细胞沉淀，用1mL PBSM洗涤沉淀，14000g离心30秒。用100μL含有Bio-OPS的PBSM缓冲液重悬酵母细胞，室温孵育1h。洗涤后，用SA-PE(BD，554061)和抗V5抗体(GenScript，A01803)对细胞进行染色，将其加入100μL PBSM缓冲液中，冰上孵育20分钟，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。经由KP19173菌株获得的Bio-OPS和/或经由KP19213菌株获得的Bio-OPS指导的筛选分别重复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，获得一系列阳性scFv抗体，并对其进行测序。
实施例2：制备和表征全长的抗O1抗体
制备全长的抗O1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)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5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50mM PBS缓冲液(包含0.15M NaCl，pH7.2)以150cm/h的流速平衡蛋白A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
将制备的全长抗O1单克隆抗体(重构成人IgG1形式)与实施例1中制备的LPS(分别经由KP19173菌株或KP19213菌株获得)进行结合试验，该试验用于鉴定结合肺炎克雷伯菌LPS的抗体，同时设置KPN70、G3-78以及MPG196为对照抗体。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1小时。随后用PBST溶液洗涤5次。向各孔中加入100μL二抗(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EC 50值。
结果表5表明：抗O1抗体G1-G7抗体以及对照抗体MPG196既可以结合KP19173菌株LPS，又可以结合KP19213菌株LPS。与对照抗体MPG196相比，抗O1抗体G1-G7抗体结合KP19173菌株或KP19213菌株LPS的能力更优或与之相当。而对照抗体KPN70仅可结合KP19173菌株LPS，对照抗体G3-78仅可结合KP19213菌株LPS。
表5
Figure PCTCN2022114384-appb-000008
Figure PCTCN2022114384-appb-000009
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，其可通过QUANTI-Blue TM底物转化率来测定。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；之后将实施例1中制备的10μL LPS(10μg/mL，来源于KP19173菌株或KP19213菌株)和起始浓度为1mg/mL的抗O1抗体(重构成人IgG1形式)梯度稀释液预先混合，加入96孔培养板中，37℃、5％CO 2孵育6-16小时。然后，将40μL上清与160μL预热的QUANTI-Blue TM(Invivogen)溶液混合，孵育60-90分钟，读取620-655nm处的吸光值。同时设置KPN70、G3-78以及MPG196为对照抗体。将仅LPS刺激和无LPS刺激时的抑制率分别计为0％和100％，归一化实验数据，计算不同浓度下抗O1抗体以及对照抗体的抑制率，经Graphpad Prism生成抑制曲线，计算IC 50值。
结果如表6所示：抗O1抗体G1-G7抗体以及对照抗体MPG196对KP19173菌株和KP19213菌株LPS均有中和活性。与对照抗体MPG196相比，抗O1抗体G1-G7抗体对KP19173菌株或KP19213菌株LPS的中和活性更优或与之相当。而对照抗体KPN70仅对KP19173菌株LPS有中和活性；对照抗体G3-78仅对KP19213菌株LPS有中和活性。与对照抗体KPN70相比，抗O1抗体G1-G7抗体中和KP19173菌株LPS的效果与之相当。与对照抗体G3-78相比，抗O1抗体G1-G7抗体展示出与之相当的中和KP19213菌株LPS的效果。
表6
Figure PCTCN2022114384-appb-000010
Figure PCTCN2022114384-appb-000011
表征全长抗O1抗体结合亲和力以及解离常数(Kd)
采用Biacore 3000(GE)表征全长抗O1抗体G2、G7(重构成人IgG1形式)以及对照抗体MPG196、KPN70与肺炎克雷伯菌O-PS的结合亲和力。将实施例1中制备的Bio-OPS(来自KP19173菌株或KP19213菌株)包被在链霉亲和素芯片(streptavidin chip)表面，检测不同浓度下的抗体与Bio-OPS的亲和力。简言之，将起始浓度为50μg/mL的抗体按照1：4倍比稀释，分别获得7个浓度梯度。结合时间设为120s，解离时间设为80s，流速30μL/min。用SPR技术测量抗体的结合速率和解离速率，并确定结合亲和力。
结果如表7所示，抗O1抗体G2或G7对于KP19173菌株或KP19213菌株O-PS均具有很高的结合亲和力。其中，与对照抗体MPG196相比，G7与肺炎克雷伯菌KP19173菌株或KP19213菌株O-PS的亲和力均提高了近10倍，与对照抗体KPN70相比则分别提高了近50倍和600倍。
抗O1抗体G2与肺炎克雷伯菌KP19173菌株或KP19213菌株O-PS的亲和力均与对照抗体MPG196相当，且分别是对照抗体KPN70的近2倍和20倍。
表7
Figure PCTCN2022114384-appb-000012
实施例3：抗O1抗体的特异性
肺炎克雷伯菌O1血清型和O2血清型O抗原中含有相同的半乳聚糖重复单元，易出现交叉反应。设计以下试验检测抗O1抗体与肺炎克雷伯菌O1血清型结合的特异性：KP19180和KP19203是两种肺炎克雷伯菌O2血清型菌株，分别代表OPS中包含不同半乳糖重复单元的O2血清型菌株。通过实施例2中所述的ELISA结合试验，检测抗O1抗体G2、G7或对照抗体MPG196、G3-78与 肺炎克雷伯菌O2血清型LPS是否结合，进而判断抗O1抗体G2、G7是否与肺炎克雷伯菌O1血清型特异性地结合。
结果所示，抗O1抗体G2、G7、对照抗体MPG196及G3-78不与O2血清型菌株KP19180的LPS结合(图2A)；抗O1抗体G2、G7、对照抗体MPG196不与KP19203的LPS结合，而对照抗体G3-78可与KP19203菌株LPS结合(图2B)。结果初步表明，本申请中的抗O1抗体G2、G7与肺炎克雷伯菌O1血清型特异性地结合，与肺炎克雷伯菌O2血清型无交叉反应。
实施例4：抗O1抗体介导的调理吞噬杀伤活性
通过调理吞噬杀伤(opsonophagocytic killing，OPK)试验确定全长抗O1抗体G2或G7(重构成人IgG1形式)以及对照抗体MPG196、G3-78介导的杀菌活性。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(KP19173菌株)中，构建发光肺炎克雷伯菌。通过检测发光肺炎克雷伯菌的相对光单位(RLU)来确定各抗体介导的杀伤活性。OPK的检测方法如文献(例如，参见DiGiandomenico,A.,et al.,InfectImmun 72,7012-7021(2004))中所述。其中，MPG196(结合Gal-II表位，因此可与KP19173结合)可作为阳性对照抗体，G3-78(结合Gal-III表位，因此不与KP19173结合)可作为阴性对照抗体。简言之，将发光肺炎克雷伯菌(KP19173菌株)、稀释的幼兔血清(Cedarlane，1:10)、由HL-60分化得到的巨噬细胞、以及不同浓度的抗体在96孔板中混合，并且在37℃、振荡(250rpm)条件下孵育2h。利用酶标仪测量相对光单位的数值。将不加入抗体和不加入肺炎克雷伯菌时测定得到的相对光单位数值分别设为0％和100％杀伤率，以此来计算不同浓度下抗O1抗体G2、G7以及对照抗体MPG196、G3-78的相对杀伤率，利用Graphpad Prism软件绘图，从而确定各抗体的调理吞噬杀伤活性。
结果如图3所示：针对KP19173菌株，抗O1抗体G2或G7抗体展示出较强的促进调理吞噬杀伤的活性，且其促进OPK的效果比阳性对照抗体MPG196更优或与之相当，而阴性对照抗体G3-78未显示出促进OPK的活性。
实施例5：抗O1抗体介导的血清杀菌活性
通过血清杀菌活性(serum bactericidial assay,SBA)试验确定全长抗O1抗体G2或G7(重构成人IgG1形式)以及对照抗体KPN70介导的补体依赖性杀菌活性。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KP19173菌株中，构建发光肺炎克雷伯菌。通过检测发光肺炎克雷伯菌的相对光单位(RLU)，确定抗O1抗体G2、G7(重构成人IgG1形式)以及对照抗体KPN70介导的补体依赖性杀菌活性。SBA的检测方法如文献(例如，参见Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils，mBio.2018Mar-Apr；9(2):e00297-18.)中所述。简言之，将发光肺炎克雷伯菌KP19173菌株和起始浓度为1.3nM的抗体梯度稀释液预先混合，加入96孔培养板中，37℃、5％CO 2孵育15min。加入25μL稀释的幼兔血清和35μL PBS缓冲液后，在37℃、振荡(250rpm)条件下孵育3h。然后利用酶标仪测量相对光单位。将不加入抗体和不加入肺炎克雷伯菌时测定得到的相对光单位数值分别 设定为0％和100％杀伤率，以此来计算不同浓度下抗O1抗体G2、G7以及对照抗体KPN70的相对杀伤率，利用Graphpad Prism软件绘图，确定各抗体的血清杀菌活性。
结果如图4A-4B所示：针对KP19173菌株，抗O1抗体G2(图4A)或G7(图4B)抗体可以诱导补体依赖的血清杀菌活性，而对照抗体KPN70未展示出补体依赖的血清杀菌活性。
实施例6：抗O1抗体结合肺炎克雷伯菌的广谱性
采用实施例2中所述的ELISA结合试验，检测抗O1抗体G2、G7与来自临床分离的其他肺炎克雷伯菌O1血清型菌株(KP1953、KP1961、KP1962或KP1963菌株)的LPS的结合情况，以判断抗O1抗体G2或G7对肺炎克雷伯菌O1血清型菌株的结合是否具有广谱性。
结果所示，抗O1抗体G2、G7均可以与来自其他O1血清型肺炎克雷伯菌菌株KP1953(图5A)、KP1961(图5B)、KP1962(图5C)或KP1963(图5D)的LPS结合。由此可见，抗O1抗体G2或G7与肺炎克雷伯菌O1血清型菌株的结合具有广谱性。
实施例7：抗O1抗体在小鼠菌血症模型中的作用
首先通过腹腔注射20mg D-(+)-半乳糖胺盐酸盐(GalN)(sigma，G1639-5G)使小鼠对内毒素敏感，再通过尾静脉注射100μL菌液(KP19173或KP19213菌株)以构建菌血症模型。若注射后24小时内小鼠全部死亡，则表示造模成功，该剂量为致死剂量，并以此剂量作为后续实验中的造模剂量。
为了确定抗O1抗体G2或G7是否具有预防保护性作用，在小鼠造模前24h时，通过腹腔注射不同浓度的抗O1抗体G2、G7(重构成人IgG1形式)、阳性对照抗体MPG196或阴性对照抗体HIV抗体HIV-10E8(Broad and potent neutralization of HIV-1 by a gp41-specific human antibody Nature 491(7424),406-412(2012))。24h后腹腔注射20mg GalN，同时通过尾静脉注射致死剂量的肺炎克雷伯菌(即KP19173菌株7500CFU/只或KP19213菌株3000CFU/只)以攻击小鼠。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。
KP19173菌株诱导的菌血症模型中的结果如图6所示，与阴性对照抗体HIV-10E8相比，在采用低剂量0.006mpk的情况下，抗O1抗体G2或G7仍能有效提高小鼠的生存率和/或延长生存时间；在采用高剂量0.018mpk的情况下，抗O1抗体G2或G7显著提高小鼠的生存率，且其对小鼠的保护效果比阳性对照抗体MPG196更优或与之相当。
在KP19213菌株诱导的菌血症模型中也取得了类似的结果：在低剂量0.45mpk或高剂量2.25mpk的情况下，抗O1抗体G2或G7均能提高小鼠的生存率，其保护效果也与对照抗体MPG196相当(结果未显示)。
上述结果表明，在菌血症动物模型中，抗O1抗体G2或G7能够显著提高小鼠的生存率，具有预防保护作用。

Claims (18)

  1. 一种分离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，其包含：
    (i)V H，其包含如氨基酸序列SEQ ID NO:39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:46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:40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:47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:41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:48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:42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:49所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (v)V H，其包含如氨基酸序列SEQ ID NO:43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:50所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (vi)V H，其包含如氨基酸序列SEQ ID NO:44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:51所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3；
    (vii)V H，其包含如氨基酸序列SEQ ID NO:45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:52所示的V L包含的LC-CDR1、LC-CDR2和LC-CDR3。
  2. 一种分离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，其包含：
    (i)V H，其包含：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:7，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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:18，LC-CDR2，其包含氨基酸序列SEQ ID NO:25，和LC-CDR3，其包含氨基酸序列SEQ ID NO:32，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，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:2，HC-CDR2，其包含氨基酸序列SEQ ID NO:8，和HC-CDR3，其包含氨基酸序列SEQ ID NO:14，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:19，LC-CDR2，其包含氨基酸序列SEQ ID NO:26，和LC-CDR3，其包含氨基酸序列SEQ ID NO:33，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，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:20，LC-CDR2，其包含氨基酸序列SEQ ID NO:27，和LC-CDR3，其包含氨基酸序列SEQ ID NO:34，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，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:4，HC-CDR2，其包含氨基酸序列SEQ ID NO:10，和HC-CDR3，其包含氨基酸序列SEQ ID NO:13，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:21，LC-CDR2，其包含氨基酸序列SEQ ID NO:28，和LC-CDR3，其包含氨基酸序列SEQ ID NO:35，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；
    (v)V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:3，HC-CDR2，其包含氨基酸序列SEQ ID NO:9，和HC-CDR3，其包含氨基酸序列SEQ ID NO:15，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:22，LC-CDR2，其包含氨基酸序列SEQ ID NO:29，和LC-CDR3，其包含氨基酸序列SEQ ID NO:36，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；
    (vi)V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:5，HC-CDR2，其包含氨基酸序列SEQ ID NO:11，和HC-CDR3，其包含氨基酸序列SEQ ID NO:16，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:23，LC-CDR2，其包含氨基酸序列SEQ ID NO:30，和LC-CDR3，其包含氨基酸序列SEQ ID NO:37，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；
    (vii)V H，其包含：HC-CDR1，其包含氨基酸序列SEQ ID NO:6，HC-CDR2，其包含氨基酸序列SEQ ID NO:12，和HC-CDR3，其包含氨基酸序列SEQ ID NO:17，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，其包含：LC-CDR1，其包含氨基酸序列SEQ ID NO:24，LC-CDR2，其包含氨基酸序列SEQ ID NO:31，和LC-CDR3，其包含氨基酸序列SEQ ID NO:38，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O1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:39或其变体，所述变体与氨基酸序列SEQ ID NO:39具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:46或其变体，所述变体与氨基酸序列SEQ ID NO:46具有至少约90％序列同一性；
    (ii)V H，其包含氨基酸序列SEQ ID NO:40或其变体，所述变体与氨基酸序列SEQ ID NO:40具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:47或其变体，所述变体与氨基酸序列SEQ ID NO:47具有至少约90％序列同一性；
    (iii)V H，其包含氨基酸序列SEQ ID NO:41或其变体，所述变体与氨基酸序列SEQ ID NO:41具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:48或其变体，所述变体与氨基酸序列SEQ ID NO:48具有至少约90％序列同一性；
    (iv)V H，其包含氨基酸序列SEQ ID NO:42或其变体，所述变体与氨基酸序列SEQ ID NO:42具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:49或其变体，所述变体与氨基酸序列SEQ ID NO:49具有至少约90％序列同一性；
    (v)V H，其包含氨基酸序列SEQ ID NO:43或其变体，所述变体与氨基酸序列SEQ ID NO:43具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:50或其变体，所述变体与氨基酸序列SEQ ID NO:50具有至少约90％序列同一性；
    (vi)V H，其包含氨基酸序列SEQ ID NO:44或其变体，所述变体与氨基酸序列SEQ ID NO:44具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:51或其变体，所述变体与氨基酸序列SEQ ID NO:51具有至少约90％序列同一性；或
    (vii)V H，其包含氨基酸序列SEQ ID NO:45或其变体，所述变体与氨基酸序列SEQ ID NO:45具有至少约90％序列同一性；以及V L，其包含氨基酸序列SEQ ID NO:52或其变体，所述变体与氨基酸序列SEQ ID NO:52具有至少约90％序列同一性。
  4. 根据权利要求1-3中任一项分离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，其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Fc片段。
  5. 根据权利要求4中分离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，其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全长的IgG抗体。
  6. 根据权利要求5中分离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，其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全长的IgG1、IgG2、IgG3或IgG4抗体。
  7. 根据权利要求1-6中任一项分离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，其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嵌合的、人源化的或全人的抗体。
  8. 根据权利要求1-3中任一项分离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，其中所述抗原结合片段选自Fab、Fab’、F(ab)’ 2、Fab’-SH、单链抗体(scFv)、Fv片段、dAb、Fd、纳米抗体(nanobody)、双链抗体(diabody)和线性抗体。
  9. 一种核酸分子，其编码权利要求1-8中任一项所述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。
  10. 一种包含权利要求9中所述的核酸分子的载体。
  11. 一种分离的宿主细胞，其包含权利要求1-8中任一项所述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、权利要求9中所述的核酸分子或权利要求10中所述的载体。
  12. 一种制备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方法，其包含：
    a)在能有效表达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条件下培养权利要求11中所述的宿主细胞；并且
    b)从宿主细胞中获得所表达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。
  13. 一种药物组合物，其包含权利要求1-8中任一项所述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、权利要求9中所述的核酸分子、权利要求10中所述的载体、权利要求11中所述的分离的宿主细胞或由权利要求12中所述的方法制备得到的抗体或抗原结合片段，以及药学上可接受的载体。
  14. 权利要求1-8中任一项所述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、权利要求9中所述的核酸分子、权利要求10中所述的载体、权利要求11中所述的分离的宿主细胞、由权利要求12中所述的方法制备得到的抗体或抗原结合片段、或权利要求13中所述的药物组合物在制备用于治疗、预防或改善所需个体疾病或病症的药物中的用途。
  15. 一种治疗疾病或病症的方法，所述方法包括向所需个体施用有效剂量的如权利要求1-8中任一项所述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、权利要求9中所述的核酸分子、权利要求10中所述的载体、权利要求11中所述的分离的宿主细胞、由权利要求12中所述的方法制备得到的抗体或抗原结合片段、或权利要求13中所述的药物组合物。
  16. 根据权利要求14中所述的用途或权利要求15中所述的治疗方法，其中所述疾病或病症与克雷伯菌感染或定植有关，包括院内感染、机会性感染、器官移植后感染以及与克雷伯菌感染/定植相关的其他疾病或病症。
  17. 根据权利要求16中所述的用途或治疗方法，其中所述克雷伯菌是肺炎克雷伯菌、产酸克雷伯菌、植生克雷伯菌、土生克雷伯菌、解鸟氨酸克雷伯菌、肉芽肿克雷伯菌、臭鼻克雷伯菌和/或鼻硬结克雷伯菌。
  18. 根据权利要求14中所述的用途、权利要求15中所述的治疗方法或权利要求16-17中任一项所述的用途或治疗方法，其中所述疾病或病症选自肺炎、***、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/CN2022/114384 2021-08-25 2022-08-24 特异性识别肺炎克雷伯菌o1抗原的抗体及其应用 WO2023025173A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202280003347.4A CN116033922A (zh) 2021-08-25 2022-08-24 特异性识别肺炎克雷伯菌o1抗原的抗体及其应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110980272.2 2021-08-25
CN202110980272 2021-08-25

Publications (1)

Publication Number Publication Date
WO2023025173A1 true WO2023025173A1 (zh) 2023-03-02

Family

ID=85321559

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/114384 WO2023025173A1 (zh) 2021-08-25 2022-08-24 特异性识别肺炎克雷伯菌o1抗原的抗体及其应用

Country Status (2)

Country Link
CN (1) CN116033922A (zh)
WO (1) WO2023025173A1 (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107371365A (zh) * 2015-02-17 2017-11-21 阿尔萨尼斯生物科学有限责任公司 靶向克雷伯氏肺炎菌半乳聚糖类o‑抗原的抗体
US20180298084A1 (en) * 2015-10-16 2018-10-18 Arsanis Biosciences Gmbh Bactericidal monoclonal antibody targeting klebsiella pneumoniae
US20180312576A1 (en) * 2015-10-28 2018-11-01 Arsanis Biosciences Gmbh Antibodies targeting a mannan-based o-antigen of k. pneumoniae
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107371365A (zh) * 2015-02-17 2017-11-21 阿尔萨尼斯生物科学有限责任公司 靶向克雷伯氏肺炎菌半乳聚糖类o‑抗原的抗体
US20180298084A1 (en) * 2015-10-16 2018-10-18 Arsanis Biosciences Gmbh Bactericidal monoclonal antibody targeting klebsiella pneumoniae
US20180312576A1 (en) * 2015-10-28 2018-11-01 Arsanis Biosciences Gmbh Antibodies targeting a mannan-based o-antigen of k. pneumoniae
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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HELD, T. K. ET AL.: "Binding to and Opsonophagocytic Activity of O-Antigen-Specific Monoclonal Antibodies against Encapsulated and Nonencapsulated Klebsiella pneumoniae Serotype O1 Strains", INFECTION AND IMMUNITY, vol. 68, no. 5, 31 May 2000 (2000-05-31), XP055258752, DOI: 10.1128/IAI.68.5.2402-2409.2000 *
KOBAYASHI SCOTT D., ADELINE R PORTER, BRETT FREEDMAN, RUCHI PANDEY, LIANG CHEN, BARRY N KREISWIRTH, FRANK R DELEO: "Antibody-Mediated Killing of Carbapenem-Resistant ST258 Klebsiella pneumoniae by Human Neutrophils", MBIO, vol. 9, no. 2, 13 March 2018 (2018-03-13), XP093038819, DOI: 10.1128/mBio.00297-18 *

Also Published As

Publication number Publication date
CN116033922A (zh) 2023-04-28

Similar Documents

Publication Publication Date Title
JP7478807B2 (ja) シュードモナス菌ＰｃｒＶを特異的に認識する抗体及びその使用
US20230257454A1 (en) Antibodies specifically recognizing c5a and uses thereof
WO2023025173A1 (zh) 特异性识别肺炎克雷伯菌o1抗原的抗体及其应用
TWI816164B (zh) 分離的抗體或抗原結合片段、其製備方法、編碼其的核酸分子、包含其的載體、宿主細胞和藥用組合物及其用途
WO2023103788A1 (zh) 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
CN115023438B (zh) 特异性识别假单胞菌pcrv或psl抗原的抗体组合以及双特异性抗体
WO2021244421A1 (en) Antibodies specifically recognizing pseudomonas pcrv and uses thereof
WO2023186054A1 (zh) 特异性识别c5a的抗体及其应用
TW202334232A (zh) 特異性識別C5aR1之抗體及其應用
CN115210258A (zh) 特异性识别胸腺基质淋巴细胞生成素的抗体及其用途
CN115925918A (zh) 特异性识别c5a的抗体及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22860519

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE