Antibodies specifically recognizing Klebsiella pneumoniae O1 antigen and their applications

Abstract

The present application relates to antibodies or antigen-binding fragments that specifically recognize Klebsiella pneumoniae O1 antigen, as well as preparation methods and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to the Chinese patent application with application number 202110980272.2, application date 2021.08.25, and invention name “Antibodies specifically recognizing Klebsiella pneumoniae O1 antigen and their applications”, and the entire contents of the application are incorporated herein by reference.

[0003] Reference to an electronic sequence listing

[0004] The contents of the electronic sequence listing (text name: CN_202107212436_SEQLIST.xml, recording date: 2022.07.12, size: 51KB) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to antibodies or antigen-binding fragments that specifically recognize Klebsiella pneumoniae O1 antigen, as well as preparation methods and uses thereof. Background Art

[0006] Klebsiella is a gram-negative bacterium that is ubiquitous in nature. Klebsiella can be found in the environment, such as surface water, sewage, soil, and plants, and can also colonize the mucosal surfaces of mammals, such as humans, horses, or pigs (Matsen, JM et al. Applied microbiology vol. 28, 4 (1974): 672-8.; Bagley, ST. 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.). For people with weakened / impaired immunity, immunodeficiency, or long-term, heavy antibiotic use leading to dysbacteriosis, Klebsiella can enter other tissues from the mucosal surface and cause diseases such as pneumonia, meningitis, liver abscess, urinary tract inflammation, 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, JA 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.). Klebsiella infections are mainly caused by Klebsiella pneumoniae, which is the most important species in the genus Klebsiella in medicine (Podschun, R, and U Ullmann. Clinical microbiology reviews vol.11,4(1998):589-603.).In recent years, strains of Klebsiella pneumoniae have become increasingly resistant to antibiotics and the rate of resistant strains has increased year by year, making this bacterial infection very difficult to treat.

[0007] Factors associated with Klebsiella virulence include capsular polysaccharides (CPS), lipopolysaccharides (LPS), adhesion factors, siderophore systems, and other virulence factors. These virulence factors play an important role in bacterial adhesion to host cells and evasion of host immune responses / immune killing. Among them, endotoxin (LPS) plays a very important role in bacterial pathogenicity and is an important molecule that causes systemic inflammatory responses in cells. LPS is a major and essential component of the outer leaflet of the cell membrane of all Gram-negative bacteria. Although the structure of LPS varies considerably between different bacteria, it is generally composed of lipid A, core oligosaccharides, and O antigens (Paczosa, Michelle K, and Joan Mecsas. Microbiology and molecular biology reviews: MMBR vol. 80, 3 629-61. 15 Jun. 2016). Because the O antigen changes much less than CPS, some scholars believe that the O antigen of Klebsiella LPS can be used as a potential target antigen for immunotherapy, becoming an alternative to antibiotic treatment (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.). Nine different O antigens have been identified in Klebsiella, of which O1 antigen is the most common (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4 (1998): 589-603.; Hansen, DS et al. Journal of clinical microbiology vol. 37, 1 (1999): 56-62.).

[0008] Previous studies have reported that polyclonal antibodies and mouse monoclonal IgG2a antibodies against the Klebsiella pneumoniae O1 antigen have a protective effect in mouse models of Klebsiella pneumoniae infection (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.). Prior art (e.g., International Application Publications WO2017064258A1 and WO2018075375A1) also demonstrate that antibodies against the Klebsiella pneumoniae O1 antigen can not only injure or kill bacteria through opsonophagocytic killing (OPK) and / or complement-dependent killing (SBA), but also neutralize endotoxins in the blood, reducing the damage caused by Klebsiella pneumoniae to the body. Therefore, the development of new anti-LPS antibodies to supplement antibiotic therapy is crucial.

[0009] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety.

[0010] Application Overview

[0011] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO: 39 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:46 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0012] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V Lcomprising: 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 the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0013] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , 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 V L , comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 46.

[0014] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:40 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:47 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0015] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises: 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, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 19, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 26, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 33, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0016] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , 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 V L , comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 47.

[0017] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:41 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:48 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0018] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0019] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , 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 V L, comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 48.

[0020] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:42 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:49 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0021] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises: 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, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0022] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 42; and V L , comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 49.

[0023] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:43 HHC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 50 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0024] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0025] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , 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 V L , comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof, said variant having at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 50.

[0026] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:44 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:51 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0027] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H, which comprises: 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, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; 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, which comprises the amino acid sequence of SEQ ID NO: 37, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0028] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , comprising 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 V L , comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 51.

[0029] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises V as shown in the amino acid sequence SEQ ID NO:45 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:52 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0030] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, comprising: H , which comprises: 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, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L, which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0031] In some embodiments, any of the isolated antibodies that specifically bind to Klebsiella pneumoniae O1 antigens described above comprises: V H , comprising 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 V L , comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 52.

[0032] In some embodiments, the isolated antibody that specifically binds to the Klebsiella pneumoniae 01 antigen binds to the Klebsiella pneumoniae 01 antigen with a Kd value of 1 pM to 5 nM.

[0033] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, which competitively binds to Klebsiella pneumoniae with any of the above-described isolated antibodies that specifically bind to a Klebsiella pneumoniae O1 antigen. In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, which specifically binds to the same epitope as any of the above-described isolated antibodies that bind to a Klebsiella pneumoniae O1 antigen.

[0034] In some embodiments, any of the separated antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen as described above, the separated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen comprises an Fc fragment. In some embodiments, the separated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is a full-length IgG antibody. In some embodiments, the separated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is a full-length IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the separated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is chimeric, fully human, or humanized. In some embodiments, the separated antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is an antigen-binding fragment selected from Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody), and linear antibody.

[0035] In some embodiments, an isolated antibody that specifically binds to a Klebsiella pneumoniae O1 antigen is provided, wherein the antibody, in addition to binding to the Klebsiella pneumoniae O1 antigen, also binds to an O1 antigen of Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinelyticus, Klebsiella granulomatosa, Klebsiella fetiferus, and / or Klebsiella rhinosclerosis.

[0036] In some embodiments, an isolated nucleic acid molecule is provided, the nucleic acid molecule encoding any of the antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen as described above. In some embodiments, a vector is provided, the vector comprising any of the nucleic acid molecules as described above. In some embodiments, a host cell is provided, the host cell comprising any of the antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen as described above, any of the nucleic acid molecules as described above, or any of the vectors as described above. In some embodiments, a method for preparing an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is provided, comprising: a) culturing any of the host cells described above under conditions effective to express an antibody that specifically binds to the Klebsiella pneumoniae O1 antigen; and b) obtaining the expressed antibody that specifically binds to the Klebsiella pneumoniae O1 antigen from the host cell.

[0037] In some embodiments, a method for treating, preventing, or ameliorating a disease or condition in a desired individual is provided, comprising administering to the individual an effective amount of any of the antibodies described above that specifically bind to a Klebsiella pneumoniae O1 antigen. In some embodiments, use of any of the antibodies described above that specifically bind to a Klebsiella pneumoniae O1 antigen in the preparation of a pharmaceutical composition for treating, preventing, or ameliorating a disease or condition in a desired individual is provided. In some embodiments, use of any of the antibodies described above that specifically bind to a Klebsiella pneumoniae O1 antigen or a pharmaceutical composition comprising an antibody that specifically binds to a Klebsiella pneumoniae O1 antigen in the preparation of a medicament for treating, preventing, or ameliorating a disease or condition is provided. In some embodiments, the disease or condition is associated with infection or colonization with Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestrial, Klebsiella ornithine-lytica, Klebsiella granulomatosa, Klebsiella fetorhinis and / or Klebsiella rhinosclerosis), including nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases or conditions associated with infection / colonization with Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestrial, Klebsiella ornithine-lytica, Klebsiella granulomatosa, Klebsiella fetorhinis and / or Klebsiella rhinosclerosis). In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy.

[0038] Also provided are pharmaceutical compositions, kits, and manufactured products comprising any of the antibodies that specifically bind to the Klebsiella pneumoniae O1 antigen as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figures 1A-1D The results shown are schematic diagrams of the LPS structures of Klebsiella pneumoniae serotypes O1 and O2. Figure 1A The results shown are schematic diagrams of the LPS structure of Klebsiella pneumoniae serotype O1 strain KP19173; Figure 1B The results shown are schematic diagrams of the LPS structure of Klebsiella pneumoniae serotype O1 strain KP19213; Figure 1C The results shown are schematic diagrams of the LPS structure of Klebsiella pneumoniae serotype O2 strain KP19180;

[0040] Figure 1D The results shown are schematic diagrams of the LPS structure of Klebsiella pneumoniae serotype O2 strain KP19203.

[0041] Figures 2A-2B The results shown are that anti-O1 antibodies G2 or G7 do not cross-react with K. pneumoniae serotype O2. Figure 2A The results shown are that anti-O1 antibodies G2 or G7 do not bind to LPS of Klebsiella pneumoniae serotype O2 strain KP19180. Figure 2B The results shown are that anti-O1 antibodies G2 or G7 do not bind to LPS of Klebsiella pneumoniae serotype O2 strain KP19203.

[0042] Figure 3 The results shown are the opsonophagocytic killing activity mediated by anti-O1 antibodies G2 or G7.

[0043] Figures 4A-4B Results shown are serum bactericidal activity mediated by anti-O1 antibodies G2 or G7. Figure 4A The bactericidal activity of serum mediated by anti-O1 antibody G2 is Figure 4B Serum bactericidal activity mediated by anti-O1 antibody G7.

[0044] Figures 5A-5D Results shown are the binding of anti-O1 antibodies G2 or G7 to LPS of other K. pneumoniae O1 serotypes. Figure 5A Results shown are for binding of anti-O1 antibodies G2 or G7 to LPS of a clinically isolated Klebsiella pneumoniae serotype O1 strain KP1953. Figure 5B Results shown are for binding of anti-O1 antibodies G2 or G7 to LPS of a clinically isolated Klebsiella pneumoniae serotype O1 strain KP1961. Figure 5C Results shown are for binding of anti-O1 antibodies G2 or G7 to LPS of a clinically isolated Klebsiella pneumoniae serotype O1 strain KP1962. Figure 5D Results shown are for binding of anti-O1 antibodies G2 or G7 to LPS of a clinically isolated Klebsiella pneumoniae serotype O1 strain KP1963.

[0045] Figure 6 The results shown are the preventive and protective effects of anti-O1 antibodies G2 or G7 in a mouse bacteremia model.

[0046] Detailed description of this application

[0047] On the one hand, the present application provides antibody molecules (i.e., anti-O1 antibodies) that specifically bind to the Klebsiella pneumoniae O1 antigen. Through a combination of natural scFv yeast library screening and appropriately designed biochemical and biological experiments, highly effective antibody molecules capable of binding to the Klebsiella pneumoniae O1 antigen and neutralizing Klebsiella pneumoniae endotoxin (LPS) have been identified. The results presented herein demonstrate that, compared to known antibodies MPG196, KPN70, or G3-78 that bind to the Klebsiella pneumoniae O antigen, the antibodies in the present application specifically bind to the Klebsiella pneumoniae O1 serotype, and surprisingly, in various biological experiments, the antibodies in the present application have been shown to perform even better than MPG196, which also specifically binds to the Klebsiella pneumoniae O1 antigen.

[0048] The anti-O1 antibodies provided herein 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.

[0049] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises the sequence DAWIS (SEQ ID NO: 1); the HC-CDR2 comprises the sequence RIRSKTDGETREYAAPVNG (SEQ ID NO: 7); and the HC-CDR3 comprises the sequence DPQWGI (SEQ ID NO: 13); and the light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASQSVSSRHLA (SEQ ID NO: 18); a LC-CDR2 comprising the sequence GASSRAT (SEQ ID NO: 25); and a LC-CDR3 comprising the sequence QQYGSTPLT (SEQ ID NO: 32).

[0050] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V Hcomprising: 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 ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RSSQNLLHSNGYNYLD (SEQ ID NO: 19); a LC-CDR2 comprising the sequence LSSNRAS (SEQ ID NO: 26); and a LC-CDR3 comprising the sequence MQALQTPYT (SEQ ID NO: 33).

[0051] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises the sequence NFWMT (SEQ ID NO: 3); the HC-CDR2 comprises the sequence NINQGGTEGYYVDSVKG (SEQ ID NO: 9); and the HC-CDR3 comprises the sequence HHGWKYNSGWRTAFDI (SEQ ID NO: 15); and the light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASQGISNSLV (SEQ ID NO: 20); a LC-CDR2 comprising the sequence GASKLHP (SEQ ID NO: 27); and a LC-CDR3 comprising the sequence QQSGRSPYT (SEQ ID NO: 34).

[0052] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H comprising: 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 (V L ), the V LIt comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence KSSQSLLHSGGKTHFY (SEQ ID NO: 21); a LC-CDR2 comprising the sequence EVSNRFS (SEQ ID NO: 28); and a LC-CDR3 comprising the sequence MQGTHWPPT (SEQ ID NO: 35).

[0053] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises the sequence NFWMT (SEQ ID NO: 3); the HC-CDR2 comprises the sequence NINQGGTEGYYVDSVKG (SEQ ID NO: 9); and the HC-CDR3 comprises the sequence HHGWKYNSGWRTAFDI (SEQ ID NO: 15); and the light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RTSQSISTHLN (SEQ ID NO: 22); a LC-CDR2 comprising the sequence GASTLQN (SEQ ID NO: 29); and a LC-CDR3 comprising the sequence QQSYRIPYS (SEQ ID NO: 36).

[0054] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the sequence NYWMT (SEQ ID NO: 5); a HC-CDR2 comprising the sequence SINQGGSEQYYVDSLKG (SEQ ID NO: 11); and a HC-CDR3 comprising the sequence ADWMSIDH (SEQ ID NO: 16); and a light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RSSQSLVNSDGNIYLS (SEQ ID NO: 23); a LC-CDR2 comprising the sequence QVSNRDS (SEQ ID NO: 30); and a LC-CDR3 comprising the sequence MQGTHWPWT (SEQ ID NO: 37).

[0055] On the other hand, the present application provides an antibody that specifically binds to Klebsiella pneumoniae O1 antigen, wherein the anti-O1 antibody comprises: a heavy chain variable domain (VH ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises the sequence GYWMS (SEQ ID NO: 6); the HC-CDR2 comprises the sequence NIKQDGSEQYYVDSVKG (SEQ ID NO: 12); and the HC-CDR3 comprises the sequence DRGIKMGSVWYPSFDL (SEQ ID NO: 17); and the light chain variable domain (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising the sequence RASRSISNYLN (SEQ ID NO: 24); a LC-CDR2 comprising the sequence AASTLQS (SEQ ID NO: 31); and a LC-CDR3 comprising the sequence QQSYSAPRT (SEQ ID NO: 38).

[0056] Also provided are nucleic acids encoding anti-O1 antibodies, compositions comprising anti-O1 antibodies, and methods of making and using anti-O1 antibodies.

[0057] definition

[0058] As used herein, "treatment" or "treating" is a method of obtaining beneficial or desired results, including clinical results. For the purposes of this application, the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., systemic spread of pathogens), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, alleviating the disease (partially or completely), reducing the dose of one or more other drugs required to treat the disease, delaying the progression of the disease, improving or enhancing the quality of life, increasing body weight, and / or prolonging survival. At the same time, "treatment" also includes a reduction in the pathological consequences of the disease (e.g., for bacterial infection, bacterial load in tissue samples, inflammatory cell infiltration). The methods of the present application take into account any one or more aspects of these treatments.

[0059] The term "prevention" refers to preventative measures including measures to prevent the occurrence of an event or measures to reduce the risk of an event.

[0060] The term "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 typically include three hypervariable loops, known as complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having heavy chains of type α, δ, ε, γ, and μ, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0061] As used herein, the term "antigen-binding fragment" includes antibody fragments, such as diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibodies (scFv), scFv dimers (divalent diabodies), multispecific antibodies composed of antibody fragments comprising one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, divalent domain antibodies, or any other antibody fragment that can bind to an antigen but does not contain a complete antibody structure. Antigen-binding fragments can bind to the same antigen as a parent antibody or parent antibody fragment (such as a parent scFv). Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments. In some embodiments, an antigen-binding fragment may include one or more CDRs from a specific human antibody that are transplanted to a framework region from one or more different human antibodies.

[0062] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. An epitope can be composed 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 exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0063] As used herein, a first antibody "competes" for binding to a K. pneumoniae 01 antigen target with a second antibody when the first antibody, at equimolar concentrations, inhibits binding of the second antibody to the K. pneumoniae 01 antigen target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%), and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0064] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as the binding of an antibody to a target that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody can specifically recognize a target (which can be an epitope) if the antibody binds to the target with greater affinity, avidity, greater ease, and / or greater persistence than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0065] As used herein, an "isolated" anti-O1 antibody is an anti-O1 antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not occur in nature.

[0066] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" is (1) unrelated to all or part of the polynucleotide sequence found in nature, (2) operably linked to a polynucleotide sequence with which it is not naturally associated, or (3) not found in nature as part of a longer sequence.

[0067] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable domains of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabatet al., USDept. 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. 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 MPet These specific regions are described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody, grafted antibody, or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the various references cited above for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this article.

[0068] Table 1: CDR definitions

[0069] <![CDATA[Kabat 1 ]]> <![CDATA[Chothia 2 ]]> <![CDATA[MacCallum 3 ]]> <![CDATA[IMGT 4 ]]> <![CDATA[AHo 5 ]]> <![CDATA[V H CDR1]]> 31-35 26-32 30-35 27-38 25-40 <![CDATA[V H CDR2]]> 50-65 53-55 47-58 56-65 58-77 <![CDATA[V H CDR3]]> 95-102 96-101 93-101 105-117 109-137 <![CDATA[V L CDR1]]> 24-34 26-32 30-36 27-38 25-40 <![CDATA[V L CDR2]]> 50-56 50-52 46-55 56-65 58-77 <![CDATA[V L CDR3]]> 89-97 91-96 89-96 105-117 109-137

[0070] 1 The amino acid residue numbers refer to the nomenclature of Kabat et al.

[0071] 2 The amino acid residue numbers refer to the nomenclature of Chothia et al.

[0072] 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al.

[0073] 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al.

[0074] 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0075] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0076] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable domain and a light chain variable domain tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and confer specificity for binding to the antibody. However, even a single variable domain (or half of an Fv fragment, which only contains 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0077] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V LThe linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0078] The term "diabodies" is used in conjunction with H and V L A small antibody fragment is prepared by constructing scFv fragments (see above) using short linkers (e.g., 5-10 residues) between the domains. This allows the variable domains to pair between chains rather than within the chain, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" scFv fragments, in which the V domains of the two antibodies are H and V L The domains 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).

[0079] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human mammal with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced by corresponding non-human residues. In addition, a humanized antibody may include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will comprise substantially all, at least one, and usually two variable domains, wherein all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0080] The "percent (%) amino acid sequence identity" or "homology" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues between a candidate sequence and the polypeptide sequence being compared, when the sequences are aligned, considering conservative substitutions as part of the sequence identity. Percent amino acid sequence identity can be determined by various alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to maximize alignment over the full length of the sequences being compared. However, for the purposes of this article, percent amino acid sequence identity values are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0081] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0082] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are reviewed in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in their intact form within and between cells, protecting them from the default degradation pathway.

[0083] The "CH1 domain" of a human IgG Fc region typically stretches from amino acid 118 to amino acid 215 (EU numbering system).

[0084] The "hinge region" is generally defined as stretching from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG isotypes can be aligned with the IgG1 sequence.

[0085] The "CH2 domain" of the human IgG Fc region typically extends from amino acids 231 to 340. The CH2 domain is unique in that it does not closely pair with another domain. Instead, two N-terminally linked branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is hypothesized that carbohydrates may serve as a substitute for domain-to-domain pairing, helping to maintain CH2 domain stability. Burton, Molec. Immunol. 22:161-206 (1985).

[0086] The "CH3" domain includes the CH2 domain extending from the C-terminal residue within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 in IgG).

[0087] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0088] IgG Fc variant antibodies with "altered" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to FcRs have a higher binding affinity (e.g., lower apparent Kd or IC) to at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. 50In some embodiments, the binding capacity is enhanced by 3-fold, e.g., 5-, 10-, 25-, 50-, 60-, 100-, 150-, 200-, or even up to 500-fold, or the binding capacity is increased by 25% to 1000%, compared to the parent polypeptide. An Fc variant that exhibits "reduced binding" to an FcR has a lower affinity for at least one FcR (e.g., a higher apparent Kd or IC) than the parent polypeptide. 50 Compared with the parent polypeptide, its binding capacity is reduced by 40% or more.

[0089] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0090] Polypeptides comprising Fc region variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. When tested in substantially the same quantity as polypeptides comprising wild-type IgG Fc polypeptides (or parent polypeptides), such polypeptides comprising Fc region variants are capable of more effectively mediating ADCC, both in vitro and in vivo. Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, such as in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0091] "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 an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased Clq binding capacity are described in U.S. Patent No. 6,194,551 Bl 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).

[0092] "Potency" is a measure of the pharmacological activity of a compound, expressed as the amount required to produce an effect of a given magnitude. 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.

[0093] "Opsonophagocytic killing" or "OPK" refers to the death of cells (eg, K. pneumoniae) that occurs due to phagocytosis by immune cells. Methods for detecting OPK activity are known in the art, see, for example, DiGiandomenico, A., et al., Infect Immun 72, 7012-7021 (2004).

[0094] Killing can also be measured using a "serum bactericidal assay" or "SBA." "SBA" also refers to the lysis of target cells by complement activation and the formation of a "membrane attack complex" in the presence of complement. Methods for evaluating the efficacy of antibodies in killing target cells using SBA assays are known in the art, see, for example, 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.

[0095] The term "Klebsiella infection" is understood as follows: Klebsiella is a Gram-negative bacterium that is a member of the Enterobacteriaceae family. It is a ubiquitous bacterium that can also colonize the human host, typically in the intestinal tract or upper respiratory tract. As an opportunistic pathogen, it can invade sterile sites from these sites if the immune system does not have proper control over it. Uncontrolled bacterial replication at these sites will induce inflammation, which is largely mediated by endotoxin (LPS) molecules released from Klebsiella. In the case of bacteremia, endotoxin molecules can trigger septic shock.

[0096] "Klebsiella colonization" means a subject has a sufficiently high concentration of Klebsiella bacteria at a site for detection, but the bacteria do not cause any signs or symptoms. Colonization can persist for a long time and is affected by the body's immune response to the organism, competition from other organisms, and sometimes the use of antimicrobial agents, which cause it to lyse (break down).

[0097] The term "neutralization" or "neutralization" broadly refers to any molecule that inhibits a pathogen from exerting its biological activity, regardless of the mechanism by which neutralization is achieved, such as inhibiting Klebsiella (e.g., Klebsiella pneumoniae) from infecting a host, or inhibiting the pathogen from promoting infection by producing endotoxins, or inhibiting endotoxins. For example, neutralization can be achieved by antibodies inhibiting Klebsiella (e.g., Klebsiella pneumoniae) from colonizing host mucosal surfaces, invading sterile body sites, and inducing adverse biological signals (in the worst case, inducing septic shock).

[0098] In a strict sense, neutralization refers to the inhibition of specific LPS binding to its cognate receptor (e.g., the Toll-like receptor-4 complex), thereby inhibiting its biological activity. Such neutralization efficacy is generally determined using standard assays known in the art, such as in vitro or in vivo neutralization assays, such as the LAL assay or TLR-4 reporter cell line assays. In these assays, for example, colorimetric assays are used to determine the inhibition of endotoxic biological activity.

[0099] Antibodies that resist or neutralize Klebsiella (e.g., Klebsiella pneumoniae) interfere with pathogens and pathogenic responses, thereby limiting or preventing infection, and / or improving the disease state caused by such infection, or inhibiting Klebsiella (e.g., Klebsiella pneumoniae) pathogenesis, particularly inhibiting the spread and replication of Klebsiella (e.g., Klebsiella pneumoniae) into the sterile body cavity / body part of the host or inhibiting its spread and replication within the sterile body cavity / body part of the host. In this regard, neutralizing antibodies can also be understood as "protective antibodies," which refer to antibodies that elicit an immune response to a pathogen observed in active or passive immunization. The neutralizing or protective antibodies described herein can be used for therapeutic purposes, such as for prevention or treatment, i.e., to prevent, improve, treat, or at least partially inhibit pathogen-induced disease symptoms, side effects, or progression. Protective antibodies can, after therapeutic application (i.e., applied to an existing infection), kill or prevent the replication of live Klebsiella (e.g., Klebsiella pneumoniae) cells, or remove all bacterial cells or their LPS molecules from sterile in vivo sites, for example, by inducing serum bactericidal or opsonophagocytic activity. Protective antibodies administered prophylactically may prevent infection from occurring through the mechanisms mentioned above or others (eg, preventing the spread of K. pneumoniae from a non-sterile site to a sterile body cavity).

[0100] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0101] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, 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. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0102] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, adenine is present at the same position in two DNA molecules, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to 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 between the two sequences is 60%. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0103] An "effective amount" of an anti-O1 antibody or composition disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods relevant to the stated purpose.

[0104] The term "therapeutically effective amount" refers to an amount or activity of an anti-O1 antibody or composition thereof disclosed herein that is sufficient to induce a beneficial effect or desired outcome (including a clinical outcome) when administered to a subject. Thus, the effective amount or synonyms depend on the context in which it is administered. An effective amount is intended to mean an amount of an antibody or composition thereof sufficient to treat, prevent, or inhibit such disease or condition. In the context of a disease, the therapeutically effective amount of the antibodies described herein is particularly useful for treating, modulating, attenuating, reversing, or affecting a disease or condition that benefits from inhibiting Klebsiella (e.g., K. pneumoniae) pathogenesis, such as adhesion and colonization of mucosal surfaces, uncontrolled replication within sterile sites, and toxicity of bacterial products to host cells. In the case of infection, the anti-O1 antibodies or compositions disclosed herein are capable of inhibiting Klebsiella (e.g., K. pneumoniae) growth and / or killing Klebsiella (e.g., K. pneumoniae). The anti-O1 antibodies may be cytostatic and / or cytotoxic. In some embodiments, a therapeutically effective amount is an amount that inhibits infection in a patient. In some embodiments, a therapeutically effective amount is an amount that completely eliminates infection in a patient.

[0105] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0106]

[0066] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0107] Reference herein to "about" for a value or parameter includes (and describes) variations of the value or parameter itself. For example, a description involving "about X" includes a description of "X".

[0108] As used herein, reference to "not" a value or parameter generally indicates and describes "other than" a value or parameter. For example, "the method cannot be used to treat type X cancer" means that the method is generally used to treat other types of cancer besides type X cancer.

[0109] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0110] Anti-O1 antibodies

[0111] In one aspect, the present application provides anti-O1 antibodies that specifically bind to Klebsiella pneumoniae O1 antigens. The anti-O1 antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs as described herein. In one aspect, the present application provides isolated antibodies that bind to Klebsiella pneumoniae O1 serotypes. Contemplated anti-O1 antibodies include, for example, full-length anti-O1 antibodies (such as full-length IgG1 or IgG4), anti-O1 single-chain antibodies, anti-O1 Fc fusion proteins, multispecific (such as bispecific) anti-O1 antibodies, anti-O1 antibody-immunoconjugates, and the like. In some embodiments, the anti-O1 antibody is a full-length antibody (such as full-length IgG1 or IgG4) or an antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O1 antigens. In some embodiments, the anti-O1 antibody is a Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, double-chain antibody (diabody) or linear antibody. In some embodiments, the antibody that specifically binds to the Klebsiella pneumoniae O1 antigen is an antibody that binds to the Klebsiella pneumoniae O1 antigen with an affinity that is at least 10 times greater than the affinity for binding to a non-target (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7In some embodiments, the anti-O1 antibody binds to an O1 antigen of Klebsiella pneumoniae, in addition to an O1 antigen of Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomata, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis. In some embodiments, a non-target antigen is not an antigen of Klebsiella genus O1. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation assay (RIA). Kd values can be determined by methods known in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0112] Although anti-O1 antibodies comprising human sequences (e.g., human heavy and light chain variable domains comprising human CDR sequences) are generally discussed herein, non-human anti-O1 antibodies are also contemplated. In some embodiments, non-human anti-O1 antibodies comprise human CDR sequences of anti-O1 antibodies described herein and non-human framework region sequences. In some embodiments, non-human framework region sequences include any sequence used to generate heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., small apes, macaques), etc. In some embodiments, non-human anti-O1 antibodies include anti-O1 antibodies generated by grafting one or more human CDR sequences described herein into non-human framework regions (e.g., murine or chicken framework region sequences).

[0113] In some embodiments, the anti-O1 antibodies described herein specifically recognize Klebsiella pneumoniae O1 antigens. In some embodiments, the anti-O1 antibodies are specific for Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomata, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis) and do not cross-react with other genera or non-O1 serotypes of Klebsiella.

[0114] In some embodiments, as described herein, any anti-O1 antibody comprises an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-O1 antibody comprises an IgG1 heavy chain constant region. In some embodiments, the anti-O1 antibody comprises an IgG2 heavy chain constant region. In some embodiments, the anti-O1 antibody comprises an IgG3 heavy chain constant region. In some embodiments, the anti-O1 antibody comprises an IgG4 heavy chain constant region. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 54. In some embodiments, the anti-O1 antibody comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 55. In some embodiments, the anti-O1 antibody comprises a lambda light chain constant region. In some embodiments, the light chain constant region comprises (consists of, or consists essentially of) the amino acid sequence of SEQ ID NO: 56. In some embodiments, the anti-O1 antibody comprises an antibody heavy chain variable domain and an antibody light chain variable domain.

[0115] In some embodiments, the anti-O1 antibody comprises: 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the 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: 32, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0116] In some embodiments, the anti-O1 antibody comprises: 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; and VL , the V L It comprises: 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.

[0117] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO: 39.

[0118] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:46.

[0119] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO: 39 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:46 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0120] In some embodiments, the anti-O1 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO: 39 or a variant thereof that has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) similarity to the amino acid sequence of SEQ ID NO: 39; and V L , comprising the amino acid sequence of SEQ ID NO: 46 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 to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 39, and V L , the V L Comprising the amino acid sequence SEQ ID NO:46.

[0121] In some embodiments, the anti-O1 antibody comprises: VH , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 19, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 26, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 33, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0122] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:19, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO:26, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO:33.

[0123] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:40.

[0124] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:47.

[0125] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:40 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:47 LContains LC-CDR1, LC-CDR2 and LC-CDR3.

[0126] In some embodiments, the anti-O1 antibody comprises: V H , 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 to the amino acid sequence of SEQ ID NO:40; and V L , comprising the amino acid sequence of SEQ ID NO: 47 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 to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 40, and V L , the V L Comprising the amino acid sequence SEQ ID NO:47.

[0127] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0128] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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 V L, which 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.

[0129] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:41.

[0130] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:48.

[0131] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:41 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:48 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0132] In some embodiments, the anti-O1 antibody comprises: V H , comprising the amino acid sequence of SEQ ID NO:41 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 to the amino acid sequence of SEQ ID NO:41; and V L , comprising the amino acid sequence of SEQ ID NO: 48 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 to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 41, and V L , the V L Comprising the amino acid sequence SEQ ID NO:48.

[0133] In some embodiments, the anti-O1 antibody comprises: V H , the V Hcomprising: 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0134] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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; and V L , which 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.

[0135] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:42.

[0136] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:49.

[0137] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:42 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:49 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0138] In some embodiments, the anti-O1 antibody comprises: V H , 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 to the amino acid sequence of SEQ ID NO:42; and V L , comprising the amino acid sequence of SEQ ID NO: 49 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 to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 42, and V L , the V L Comprising the amino acid sequence SEQ ID NO:49.

[0139] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0140] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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 V L, which 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.

[0141] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:43.

[0142] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:50.

[0143] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:43 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO: 50 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0144] In some embodiments, the anti-O1 antibody comprises: V H , 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 to the amino acid sequence of SEQ ID NO:43; and V L , comprising the amino acid sequence of SEQ ID NO: 50 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 to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 43, and V L , the V L Comprising the amino acid sequence SEQ ID NO:50.

[0145] In some embodiments, the anti-O1 antibody comprises: V H , the V Hcomprising: 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; 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, which comprises the amino acid sequence of SEQ ID NO: 37, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0146] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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; 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, which comprises the amino acid sequence of SEQ ID NO:37.

[0147] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:44.

[0148] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:51.

[0149] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:44 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:51 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0150] In some embodiments, the anti-O1 antibody comprises: V H , 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 to the amino acid sequence of SEQ ID NO: 44; and V L , comprising the amino acid sequence of SEQ ID NO: 51 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 to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 44, and V L , the V L Comprising the amino acid sequence SEQ ID NO:51.

[0151] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0152] In some embodiments, the anti-O1 antibody comprises: V H , the V H 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; and V L, which 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.

[0153] In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising one, two or three HC-CDRs of the amino acid sequence of SEQ ID NO:45.

[0154] In some embodiments, the anti-O1 antibody comprises: V L , the V L Comprising one, two or three LC-CDRs of the amino acid sequence of SEQ ID NO:52.

[0155] In some embodiments, the anti-O1 antibody comprises: V H , which comprises V as shown in the amino acid sequence SEQ ID NO:45 H HC-CDR1, HC-CDR2 and HC-CDR3 included; and V L , which comprises V as shown in the amino acid sequence SEQ ID NO:52 L Contains LC-CDR1, LC-CDR2 and LC-CDR3.

[0156] In some embodiments, the anti-O1 antibody comprises: V H , 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 to the amino acid sequence of SEQ ID NO: 45; and V L , comprising the amino acid sequence of SEQ ID NO: 52 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 to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the anti-O1 antibody comprises: V H , the V H Comprising the amino acid sequence SEQ ID NO: 45, and V L , the V L Comprising the amino acid sequence SEQ ID NO:52.

[0157] In some embodiments, the above-mentioned amino acid substitutions are limited to the "exemplary substitutions" shown herein in Table 4. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown herein in Table 4.

[0158] In some embodiments, the present application provides antibodies that compete with any of the anti-O1 antibodies described herein for binding to the Klebsiella pneumoniae O1 antigen. In some embodiments, antibodies that can compete with any of the anti-O1 antibodies described herein for binding to the Klebsiella pneumoniae O1 antigen are provided. In some embodiments, anti-O1 antibodies are provided that are combined with a V H and V L The anti-O1 antibody molecules bind to the same epitope, wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 39-45, and said V L In some embodiments, an anti-O1 antibody is provided, which is combined with a V H and V L The anti-O1 antibody competitively binds to the Klebsiella pneumoniae O1 antigen, wherein the V H comprising the amino acid sequence shown in any one of SEQ ID NOs: 39-45, and said V L Comprising the amino acid sequence shown in any one of SEQ ID NOs:46-52.

[0159] In some embodiments, competition assays can be used to identify monoclonal antibodies that compete with the anti-O1 antibodies described herein for binding to the K. pneumoniae O1 antigen. Competition assays can determine whether two antibodies bind to the same epitope by recognizing the same or spatially overlapping epitopes or by competitively inhibiting binding of one 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 such as those described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody. In some embodiments, the antibody that competes with the anti-O1 antibodies described herein is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0160] Exemplary anti-O1 antibody sequences are shown in Tables 2 and 3, where CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms (Kabat definition) for predicting the positions of CDRs and defining the variable regions of antibody light and heavy chains. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, are also within the scope of this application.

[0161] Table 2 Exemplary anti-O1 antibody CDR sequences

[0162]

[0163]

[0164] Table 3 Exemplary sequences

[0165]

[0166]

[0167] Klebsiella

[0168] The vast majority of Klebsiella infections are associated with hospitalization. As a conditional pathogen, Klebsiella primarily attacks individuals with severe underlying diseases (such as diabetes or chronic pulmonary obstructive pulmonary disease) and weakened immune systems. Over time and as taxonomy continues to evolve, the classification of Klebsiella has been revised, resulting in three main classifications: Cowan, Bascomb, and in Klebsiella is classified into five categories, including Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella terrestris, Klebsiella vegetative and Klebsiella ornithine-lytica, and Klebsiella pneumoniae also includes Klebsiella pneumoniae with odor and Klebsiella pneumoniae with rhinosclerosis (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4 (1998): 589-603.). In addition, Klebsiella granulomata, etc., which appear according to other classification / nomenclature methods, are also considered within the scope of this application. Medically, Klebsiella pneumoniae is the species that causes the most infections and is also the most important species in the genus Klebsiella. Klebsiella pneumoniae can cause diseases such as sepsis, pneumonia, urinary tract infection, rickets, etc. (Podschun, R, and U Ullmann. Clinical microbiology reviews vol. 11, 4 (1998): 589-603.).

[0169] LPS

[0170] LPS is a major surface antigen, which is composed of O-specific polysaccharides (O-PS) containing different numbers of oligosaccharide repeating units (RU), core oligosaccharides and lipid A. The O-PS structure defines the O-serotype of Klebsiella strains. Compared with most Gram-negative bacteria, the variability of Klebsiella O antigen currently has only 9 major O serotypes: O1, O2, O2ac, O3, O4, O5, O7, O8, O12 (Hansen, DS et al. Journal of clinical microbiology vol. 37, 1 (1999): 56-62.) and some subtypes in these serogroups (Kelly, RF, and C Whitfield. Journal of bacteriology vol. 178, 17 (1996): 5205-14.). According to published epidemiological data, serotypes O1 and O2 account for 50-68% of all Klebsiella infections (Hansen, DS 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). The LPS expressed by O1 and O2 strains contains O-PS composed of galactose (gal) homopolymers. The O1 serotype expresses D-galactan-I (gal-I), which is composed of a repeating unit of →3)-β-D-Galf-(1→3)-α-D-Galp-(1→) and D-galactan-II (gal-II), which is composed of a repeating unit of →3)-α-D-Galp-(1→3)-β-D-Galp-(1→) (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 a structure unique to O1 type LPS (Pennini, Meghan E et al. Nature communications vol. 8, 1 1991. 8 Dec. 2017). On the other hand, O2 is composed only of gal-I (Whitfield, C et al. Journal of bacteriology vol.174,15(1992):4913-9.).For both serotypes, gal-I synthesis is encoded by the his-linked rfb (wb) operon (Clarke, BR, and C Whitfield. Journal of bacteriology vol. 174, 14 (1992): 4614-21.; Kelly, RF, and C Whitfield. Journal of bacteriology vol. 178, 17 (1996): 5205-14.). In addition, from a genetic point of view, the O1 strain carries an unlinked locus (wbbYZ) responsible for the synthesis of D-gal-II (Hsieh, Pei-Fang et al. Frontiers in microbiology vol. 5 608. 19 Nov. 2014). Previous studies have shown that D-gal-I of the O2 serotype can be modified by stoichiometric or non-stoichiometric addition of O-acetyl or terminal D-galactose (Kelly et al., 1995). Recent studies have revealed the frequent occurrence of gal-I backbone repeat units modified with terminal α-D-Galp residues in the O2 serogroup, namely (→3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→), which is called D-galactan-III (gal-III), and the genetic background of this modification has been determined (Szijártó, Valéria et al. International journal of medical microbiology: IJMM vol. 306, 2 (2016): 89-98.). The results show that the conversion of gal-I to gal-III is encoded by gmlABC, which is adjacent to the rfb (wb) operon encoding gal-I. Furthermore, studies have shown that approximately 40% of O1 clinical isolates carry the gmlABC gene (Szijártó, Valéria et al. International Journal of Medical Microbiology: IJMM vol. 306, 2 (2016): 89-98). This suggests that gal-III is also expressed within the O1 serotype. Structural analysis of extracted LPS or isolated O-PS revealed the coexistence of gal-II and gal-III. Ultimately, gal-II can combine with either gal-I or gal-III homopolymers, resulting in both scenarios in the O1 serotype.The O2 serotype is composed only of gal-I or gal-III homopolymers (Stojkovic, Katarina et al. Frontiers in microbiology vol.8 684.25 Apr.2017).

[0171] Full-length anti-O1 antibody

[0172] In some embodiments, the anti-O1 antibody is a full-length anti-O1 antibody. In some embodiments, the full-length anti-O1 antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-O1 antibody comprises an IgG constant region, such as a constant region of IgG1, IgG2, IgG3, IgG4, or variants thereof. In some embodiments, the full-length anti-O1 antibody comprises a lambda light chain constant region. In some embodiments, the full-length anti-O1 antibody comprises a kappa light chain constant region. In some embodiments, the full-length anti-O1 antibody is a full-length human anti-O1 antibody. In some embodiments, 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 modified Fc sequence such that it has altered or enhanced effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular phagocytosis (ADCP).

[0173] Thus, for example, in some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody specifically binds to a Klebsiella pneumoniae O1 antigen. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0174] In some embodiments, a full-length anti-O1 antibody comprising an IgG2 constant region is provided, wherein the anti-O1 antibody specifically binds to a Klebsiella pneumoniae O1 antigen. In some embodiments, the IgG2 is a human IgG2. In some embodiments, 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.

[0175] In some embodiments, a full-length anti-O1 antibody comprising an IgG3 constant region is provided, wherein the anti-O1 antibody specifically binds to a Klebsiella pneumoniae O1 antigen. In some embodiments, the IgG3 is a human IgG3. In some embodiments, 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.

[0176] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody specifically binds to a Klebsiella pneumoniae O1 antigen. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0177] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-12 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 13-17 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 18-24 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25-31 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 32-38 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0178] In some embodiments, a full-length anti-O1 antibody comprising an IgG2 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-12 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 13-17 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 18-24 of any one of the amino acid sequences or variants thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25-31 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 32-38 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG2 is a human IgG2. In some embodiments, 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.

[0179] In some embodiments, a full-length anti-O1 antibody comprising an IgG3 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions, an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-12 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 13-17 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 18-24 of any one of the amino acid sequences or variants thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25-31 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids; and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 32-38 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG3 is a human IgG3. In some embodiments, 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.

[0180] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; an HC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 7-12 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and an HC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 13-17 or a variant thereof, wherein the variant comprises up to about 3 (e.g., 1, 2, or 3) amino acid substitutions; and b) a light chain variable domain comprising: an LC-CDR1 comprising an amino acid sequence as shown in any one of SEQ ID NOs: NOs: 18-24 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, LC-CDR2 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 25-31 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids, and LC-CDR3 comprising an amino acid sequence as shown in any one of SEQ ID NOs: 32-38 or a variant thereof, the variant comprising a substitution of up to about 3 (e.g., 1, 2, or 3) amino acids. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0181] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-6, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7-12, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 13-17, or a variant of the heavy chain variable domain comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequence; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 18-24, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 25-31, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: NOs:32-38, or a variant of the light chain variable domain, comprising a substitution of up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acids in its LC-CDR sequence. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, 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.

[0182] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-6, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7-12, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 13-17, or a variant of the heavy chain variable domain comprising up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acid substitutions in the HC-CDR sequence; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 18-24, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 25-31, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: NOs:32-38, or a variant of the light chain variable domain, comprising a substitution of up to about 5 (e.g., 1, 2, 3, 4, or 5) amino acids in its LC-CDR sequence. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, 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.

[0183] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-6, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7-12, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 13-17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 18-24, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 25-31, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 32-38. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0184] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1-6, an HC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 7-12, and an HC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 13-17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 18-24, an LC-CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 25-31, and an LC-CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 32-38. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0185] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0186] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0187] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein 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 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. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0188] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO:10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO:13; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO:28, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO:35. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, 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.

[0189] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0190] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0191] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0192] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 32. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0193] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 19, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 26, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0194] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 20, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 34. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0195] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 10, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 13; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 35. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0196] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 3, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 9, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 15; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 22, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 29, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 36. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0197] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 5, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 23, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 37. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0198] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 6, an HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and an HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 24, an LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and an LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 38. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0199] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 39-45 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39-45; and a light chain variable domain (V L ), the V Lcomprising the amino acid sequence of any one of SEQ ID NOs: 46-52 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46-52. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0200] In some embodiments, a full-length anti-O1 antibody comprising an IgG2 constant region is provided, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 39-45 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39-45; and a light chain variable domain (V L ), the V L The invention also provides an IgG2 antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 46-52, 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 to any one of SEQ ID NOs: 46-52. In some embodiments, the IgG2 antibody is a human IgG2 antibody. In some embodiments, 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.

[0201] In some embodiments, a full-length anti-O1 antibody comprising an IgG3 constant region is provided, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V Hcomprising the amino acid sequence of any one of SEQ ID NOs: 39-45 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39-45; and a light chain variable domain (V L ), the V L The invention also provides an IgG3 antibody comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 46-52, 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 to any one of SEQ ID NOs: 46-52. In some embodiments, the IgG3 antibody is a human IgG3 antibody. In some embodiments, 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.

[0202] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: a heavy chain variable domain (V H ), the V H comprising the amino acid sequence of any one of SEQ ID NOs: 39-45 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 39-45; and a light chain variable domain (V L ), the V Lcomprising the amino acid sequence of any one of SEQ ID NOs: 46-52 or a variant thereof, wherein the variant has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of any one of SEQ ID NOs: 46-52. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0203] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs:39-45, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs:46-52. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:53. In some embodiments, 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.

[0204] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises a heavy chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs:39-45, and a light chain variable domain comprising the amino acid sequence of any one of SEQ ID NOs:46-52. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:54. In some embodiments, 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.

[0205] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 46. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0206] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V Hcomprising 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0207] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0208] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 42; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0209] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V Lcomprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 50. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0210] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0211] In some embodiments, a full-length anti-O1 antibody comprising an IgG1 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V Hcomprising 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the IgG1 is a human IgG1. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, 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.

[0212] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the 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. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0213] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 47 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 47. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0214] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V Lcomprising 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. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0215] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 42; and V L , the V L comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 49. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0216] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V Hcomprising 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 V L , the 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. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0217] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0218] In some embodiments, a full-length anti-O1 antibody comprising an IgG4 constant region is provided, wherein the anti-O1 antibody comprises: V H , the V H 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 V L , the V L comprising the amino acid sequence of SEQ ID NO: 52 or a variant thereof having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 52. In some embodiments, the IgG4 is a human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0219] Binding affinity

[0220] Binding affinity is expressed as Kd, Koff, Kon or Ka. As used herein, the term Koff refers to the rate constant for the dissociation of an antibody from an antigen / antibody complex, as measured by a kinetic selection apparatus. The term 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 for a specific antibody-antigen interaction, and is the concentration of antigen required for the antigen to occupy half of all antibody binding sites in a solution of antibody molecules and to reach equilibrium, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antibody is attached to the cell wall, such as in a yeast expression system, the corresponding equilibrium dissociation rate constant is expressed as EC 50 It is expressed as , which is a good approximation of Kd. The affinity binding constant, Ka, is the reciprocal of the dissociation constant, Kd.

[0221] The dissociation constant (Kd) can be used as an indicator of the affinity of the reactive antibody portion for the antigen. For example, a simple analysis can be performed using antibodies labeled with various markers using the Scatchard method and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values obtained using these methods are expressed in units of M. An antibody that specifically binds to a target may have, for example, a Kd of ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0222] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0223] In some embodiments, 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 (e.g. 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). Thus, in some embodiments, the Kd value for 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 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, 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.

[0224] In some embodiments, the Kd value of the anti-O1 antibody binding to the non-target is higher than the Kd value of the anti-O1 antibody binding to the target, and in some embodiments cited herein, the binding affinity of the anti-O1 antibody to the target (e.g., K. pneumoniae O1 antigen) is higher than the binding affinity of the anti-O1 antibody to the non-target. In some embodiments, the non-target is an antigen other than the K. pneumoniae O1 antigen. In some embodiments, the Kd value of the anti-O1 antibody (directed to the K. pneumoniae O1 antigen) binding to the non-K. pneumoniae O1 antigen target differs by at least 10-fold, e.g., 10-100-fold, 100-1000-fold, 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.

[0225] In some embodiments, the anti-O1 antibody binds to a non-target with a Kd value of 10 -1 M to 10 -6 M (e.g. 10 -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target is an antigen other than Klebsiella pneumoniae O1 antigen. Thus, in some embodiments, the Kd value of the 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 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0226] In some embodiments, when it is mentioned that the anti-O1 antibody specifically recognizes the Klebsiella pneumoniae O1 antigen target with high binding affinity and binds to non-targets with low binding affinity, the Kd value of the anti-O1 antibody binding to the Klebsiella pneumoniae O1 antigen target is 10 -7 M to 10 -13 M (e.g. 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), and the Kd value for binding to non-target is 10 -1 M to 10 -6 M (e.g. 10-1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0227] In some embodiments, when it is mentioned that the anti-O1 antibody specifically recognizes the Klebsiella pneumoniae O1 antigen, the binding affinity of the anti-O1 antibody is compared with the binding affinity of a control anti-O1 antibody (e.g., MPG196) or other anti-O antigen antibodies (e.g., KPN70 or G3-78). In some embodiments, the Kd value of binding between the control anti-O1 antibody or anti-O antigen antibody and the Klebsiella pneumoniae O1 antigen can be at least 2 times, 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, 1 ... 3 -10 4 times.

[0228] Nucleic Acids

[0229] Nucleic acid molecules encoding anti-O1 antibodies are also contemplated. In some embodiments, a nucleic acid (or set of nucleic acids) encoding a full-length anti-O1 antibody is provided, including any of the full-length anti-O1 antibodies described herein. In some embodiments, the nucleic acid (or set of nucleic acids) encoding the anti-O1 antibody described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His tag, an HA tag).

[0230] Also contemplated herein are isolated host cells comprising an anti-O1 antibody, isolated nucleic acids encoding an anti-O1 antibody polypeptide component, or vectors comprising nucleic acids encoding an anti-O1 antibody polypeptide component as described herein.

[0231] The present application also includes variants of these nucleic acid sequences. For example, the variant includes a nucleotide sequence that hybridizes to the nucleic acid sequence encoding the anti-O1 antibody of the present application under at least moderately stringent hybridization conditions.

[0232] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0233] Briefly, 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, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the anti-O1 antibody (e.g., a full-length anti-O1 antibody). The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate expression of the target nucleic acid sequence.

[0234] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0235] Nucleic acids can be cloned into many types of vectors. For example, 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.

[0236] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0237] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0238] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are interchanged or moved. In the thymidine kinase (Tk) promoter, activity only begins to decline when the spacing between promoter elements increases to 50 bp.

[0239] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to the use of only constitutive promoters, as inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when it is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0240] In some embodiments, expression of the anti-O1 antibody is inducible. In some embodiments, the nucleic acid sequence encoding the anti-O1 antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0241] Inducible promoter

[0242] The use of an inducible promoter provides a molecular switch that turns on the expression of an operably linked polynucleotide sequence 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-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system used to express anti-O1 antibodies is the Tet system. In some embodiments, the inducible promoter system used to express anti-O1 antibodies is the E. coli lac repression system.

[0243] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0244] In one embodiment, 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, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0245] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0246] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transcriptional activation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0247] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0248] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0249] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and that encodes a polypeptide whose expression is expressed as some easily detectable properties, such as enzymatic activity. After the DNA is introduced into the recipient cell, the expression of the reporter gene is detected at an appropriate time. Suitable reporter genes can include genes encoding luciferase, β-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 well known and can be prepared by known techniques or obtained commercially. Generally, the construct of the minimum 5' flanking region that can show the highest expression level of the reporter gene is identified as a promoter. Such a promoter region can be connected to a reporter gene and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0250] In some embodiments, nucleic acids encoding any of the full-length anti-O1 antibodies described herein are provided. In some embodiments, the nucleic acids include one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-O1 antibodies. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some of the nucleic acid sequences are contained in the same vector. In some embodiments, all of the nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, a mammalian expression vector and a viral vector (e.g., a vector derived from a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, and a lentivirus).

[0251] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0252] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0253] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly 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, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0254] 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 micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0255] In the case of using a non-viral delivery system, an exemplary delivery vehicle is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). On the other hand, the nucleic acid can be combined with lipids. The nucleic acid combined with lipids can be wrapped into the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, connected to the liposome by a linker molecule combined with the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, be dispersed in a solution containing lipids, mix with lipids, combine with lipids, be suspended in lipids, be contained in micelles or mix with micelles, or otherwise combine with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any particular structure in solution. For example, they may exist with a bilayer structure, with micelles or with a "collapsed" structure. They can also simply be dispersed in solution and may form aggregates of uneven size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, 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, amino alcohols, and aldehydes.

[0256] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of 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, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or identification by the assays described herein, all fall within the scope of the present application.

[0257] Preparation of anti-O1 antibodies

[0258] In some embodiments, the anti-O1 antibody is a monoclonal antibody or is derived from a monoclonal antibody. In some embodiments, the anti-O1 antibody comprises a V from a monoclonal antibody. H and V L , or variants thereof. In some embodiments, the anti-O1 antibody further comprises the CH1 and CL regions from a monoclonal antibody, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, including hybridoma cell methods, phage display methods, or recombinant DNA methods. In addition, exemplary phage display methods are described herein and in the examples below.

[0259] In the hybridoma method, an immunizing agent is usually used to immunize hamsters, mice or other suitable host animals to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0260] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0261] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0262] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0263] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0264] In some embodiments, according to any of the anti-O1 antibodies described herein, the anti-O1 antibody comprises the sequence of a clone selected from an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening combinatorial libraries of antibody fragments for desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries for antibodies with desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, 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, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0265] In some phage display methods, V H and V LAll components of the gene are randomly recombined in a phage library, and then phages that can bind to the antigen are screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages usually display antibody fragments in the form of scFv fragments or Fab fragments. Library phages of immune origin provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against a variety of non-self antigens and self-antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0266] The anti-O1 antibody is prepared by screening the anti-O1 antibody portion of the library that can specifically bind to the target Klebsiella pneumoniae O1 antigen through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, 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) kinds of unique human antibody fragments with different diversity. In some embodiments, the library is a human natural library constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all heavy and light chain subfamilies. In some embodiments, 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. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 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. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0267] Phage clones with high affinity to target Klebsiella pneumonia O1 antigen can be screened by iterative binding of phage to target Klebsiella pneumonia O1 antigen, wherein the target Klebsiella pneumonia O1 antigen is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning), followed by removal of unbound phage, and elution of the specifically bound phage. Subsequently, the eluted bound phage clones are used to infect suitable host cells, such as E.coli XL1-Blue, for expression and purification. For example, multiple rounds of panning (e.g., 2, 3, 4, 5, 6 or more rounds) can be performed, such as solution panning, cell panning or both, in combination with enrichment for the phage clones that specifically bind to the Klebsiella pneumonia O1 antigen. The specific binding of the phage clones to the target Klebsiella pneumonia O1 antigen can be detected by any method known in the art, including, for example, ELISA and FACS.

[0268] Another method for screening antibody libraries is to display proteins on the surface of yeast cells. Wittrup et al. (U.S. Patents 6,699,658 and 6,696,251) developed a method for displaying libraries on yeast cells. In this yeast display system, one component includes a yeast lectin protein (Aga1) anchored to the yeast cell wall, and the other component includes the second subunit of the lectin protein Aga2, which can bind to the Aga1 protein through a disulfide bond and then be displayed on the yeast cell surface. The Aga1 protein is expressed by integrating the Aga1 gene into the yeast chromosome. A single-chain variable fragment (scFv) library is fused to the Aga2 gene in a yeast display plasmid, and after transformation, the library can be 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.

[0269] Human antibody V gene library (V H and V K The fragments were obtained by PCR using a set of degenerate primers (Sblattero, D. and Bradbury, A. Immunotechnology 3, 271-278 1998). The PCR templates were obtained from commercially available RNA or cDNA, including PBMC, spleen, lymph node, bone marrow and tonsil. H and V K After the PCR libraries were combined, they were assembled into scFv formats by overlap extension PCR (Sheets, MD et al, Proc. Natl. Acad. Sci. USA 95, 6157–6162, 1998). To construct a yeast scFv display library, the resulting scFv PCR products were 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).

[0270] Mammalian cell display systems can be used to screen for anti-O1 antibodies, in which the antibody portion is displayed on the cell surface and antibodies that specifically target the Klebsiella pneumoniae O1 antigen are isolated by antigen-directed screening methods (as described in US Patent No. 7,732,195B2). A Chinese hamster ovary (CHO) cell library displaying a large number of human IgG antibody genes can be established and used to discover clones expressing high-affinity antibody genes. Another display system has been developed that allows the same protein to be simultaneously displayed and secreted on the cell surface through alternative splicing, in which the displayed protein phenotype remains associated with the genotype, allowing the secreted soluble antibody to be characterized in both biophysical and cell-based functional assays. This method overcomes many of the limitations of previous mammalian cell display and enables direct screening and maturation of full-length, glycosylated IgGs (Peter M. Bowers, et al, Methods 2014, 65: 44-56). Transient expression systems are suitable for a single round of antigen selection before antibody gene recovery and are therefore most useful for selecting antibodies from smaller libraries. Stable exosome vectors offer an attractive alternative. Exosome vectors can be efficiently transfected and stably maintained at low copy numbers, allowing for multiple rounds of panning and the resolution of more complex antibody repertoires.

[0271] The IgG library was constructed based on the ligation of germline sequence V gene segments isolated from a group of human donors and rearranged (D)J regions. RNA collected from human blood samples was reverse transcribed into cDNA and the V H and V K Specific primers amplify V H and V K The fragment was purified by gel extraction. H and V K The fragments were subcloned into display vectors containing IgG1 or K constant regions, and then electroporated or transduced into 293T cells to prepare IgG libraries. H and V K To generate scFv, the fragments are then subcloned into a display vector and electroporated or transduced into 293T cells. As is well known, IgG libraries are constructed based on germline sequence V gene segments and rearranged (D)J regions isolated from a group of donors, which can be mice, rats, rabbits or monkeys.

[0272] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that specifically bind to the genes encoding the light and heavy chains of the murine antibody). The hybridoma cells described above or the Klebsiella pneumoniae O1 antigen-specific yeast clones of this application can be used as sources of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in recombinant host cells. The DNA can also be modified, for example, by replacing the human heavy and light chain constant regions with coding sequences and / or replacing homologous non-human sequences with framework regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking the coding sequence of an immunoglobulin to the coding sequence of a non-immunoglobulin polypeptide in whole or in part. This non-immunoglobulin polypeptide can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable domain of the antibody in the present application to form a chimeric bivalent antibody.

[0273] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0274] 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.

[0275] The antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to an immunoglobulin constant region. Preferably, the fusion is performed with an immunoglobulin heavy chain constant region, which includes at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy chain constant region (CH1) containing the necessary sites for light chain binding is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion, and if desired, DNA encoding the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism.

[0276] Fully human and humanized antibodies

[0277] The anti-O1 antibody (such as a full-length anti-O1 antibody) can be a fully human antibody or a humanized antibody. The humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding subsequences of an antibody), which generally includes minimal sequences derived from non-human immunoglobulins. Humanized antibodies include human immunoglobulins, immunoglobulin chains, or fragments thereof (recipient antibodies) in which the residues of the recipient CDRs are replaced by CDR residues of non-human (donor antibody) with the desired specificity, affinity, and performance, such as mouse, rat, or rabbit CDRs. In some embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain amino acid residues that are neither in the recipient antibody nor in the introduced CDR or framework region sequences. Generally, a humanized antibody comprises at least one, and typically two, variable domains, in which all or substantially all CDR regions correspond to those of a non-human immunoglobulin and all or substantially all framework regions are human immunoglobulin consensus sequences.

[0278] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable domain. According to some embodiments, humanization can be performed essentially according to the following method of Winter and 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 replacing the corresponding sequence of a human antibody with rodent CDRs or CDR sequences. Therefore, this "humanized" antibody portion (US Patent No. 4,816,567), which is substantially less than a complete human antibody, has its variable domains replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0279] Fully human antibodies are an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies after immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce human antibodies upon antigen stimulation, see, for example, 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); US Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that produced in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US 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 Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0280] Fully human antibodies can also be produced by in vitro activated B cells (see US 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). The techniques of Cole et al. and Boerner et al. 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).

[0281] Anti-O1 antibody variants

[0282] In some embodiments, amino acid sequences of anti-O1 antibody variants (e.g., full-length anti-O1 antibodies) provided herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological activities of the antibody. The amino acid sequence of the antibody variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody 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 construct can be achieved by any combination of deletions, insertions, and substitutions of amino acid residues to impart the desired characteristics, such as antigen binding.

[0283] In some embodiments, anti-O1 antibody variants are provided having one or more amino acid substitutions. Target sites for substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody, and the product screened for desired activity, e.g., improved biological activity, maintained / improved antigen binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0284] Conservative substitutions are shown in Table 4 below.

[0285] Table 4 Conservative substitutions

[0286] Original residue Exemplary Substitutions Preferred substitution 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

[0287] 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

[0288] Amino acids are divided into different categories based on the properties of their side chains:

[0289] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Isoleucine;

[0290] b. Neutral hydrophilic amino acids: cysteine Cys, serine Ser, threonine Thr, asparagine Asn, glutamine Gln;

[0291] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0292] d. Basic amino acids: histidine His, lysine Lys, arginine Arg;

[0293] e. Contains amino acids that affect chain direction: glycine Gly, proline Pro;

[0294] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0295] Non-conservative amino acid substitutions include substituting one class for another.

[0296] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants with specific biological activity (e.g., based on neutralization activity or binding affinity) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved neutralization activity or binding affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variant V H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0297] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 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. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0298] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0299] A useful method for identifying amino acid residues or regions in antibodies that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or additionally, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0300] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides comprising 100 or more residues, and also include insertions of one or more amino acid residues within a sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include fusions of an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody molecule to the N- or C-terminus of the antibody molecule.

[0301] Fc region variants

[0302] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., a full-length anti-O1 antibody or anti-O1 antibody fusion protein), thereby generating an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC, CDC, and / or ADCP potency, typically associated with Fc-binding receptors (FcRs). In some embodiments, the Fc region variant has reduced ADCC, CDC, and / or ADCP potency. There are many examples of how changes or mutations in Fc sequences affect their potency. For example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0303] Antibodies can be modified and / or changed in the Fc region to provide desired effector functions or serum half-life. As discussed in more detail in the following sections, in the case of having appropriate Fc regions, the naked antibodies combined on the cell surface can induce cytotoxicity in the following manner: via antibody-dependent cellular toxicity (ADCC); by raising complement in complement-dependent cytotoxicity (CDC); or by raising non-specific cytotoxic cells expressing one or more effector ligands, these effector ligands recognize the antibodies combined on bacteria (such as Klebsiella pneumoniae) and subsequently cause phagocytosis of cells in antibody-dependent cell-mediated phagocytosis (ADCP); or by other mechanisms. In the case of expecting to eliminate or reduce effector functions (such as reducing side effects or treating complications), modified and / or changed Fc regions can be used, for example, to increase binding affinity to FcRn and increase serum half-life. The Fc region can also be conjugated with moieties such as PEG or albumin to increase serum half-life.

[0304] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against diseased cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the surface of the target cell membrane are bound by specific antibodies (e.g., anti-O1 antibodies). The ADCC effect typically involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody leads to activation of the NK cell, release of cytolytic granules, and subsequent apoptosis of the target cell.

[0305] In some embodiments, the present application also provides anti-O1 antibody variants (e.g., full-length anti-O1 antibody variants) that contain an Fc region with some, but not all, effector functions, resulting in an extended half-life in vivo. However, specific effector functions (e.g., CDC or ADCC) are unnecessary or deleterious, making such anti-O1 antibodies ideal candidates for the present application. Reduction / elimination of CDC and / or ADCC activity can be confirmed by in vitro and / or in vivo cytotoxicity assays. For example, an Fc receptor (FcR) binding assay can be used to confirm that the antibody lacks FcγR binding ability (and therefore may lack ADCC activity) but still retains FcRn binding ability. Among the primary cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. 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 a molecule of interest are described in US Pat. No. 5,500,362 (see, e.g., 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); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be employed (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models 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 does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and M. J. Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12): 1759-1769 (2006)).

[0306] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0307] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0308] In some embodiments, an anti-O1 antibody (e.g., a full-length anti-O1 antibody) variant is provided, comprising an Fc region variant having one or more amino acid substitutions capable of enhancing ADCC. In some embodiments, the Fc region variant comprises one or more amino acid substitutions capable of enhancing ADCC, wherein the substitutions are at positions 298, 333, and / or 334 of the Fc region (EU residue numbering). In some embodiments, the anti-O1 antibody (e.g., a full-length anti-O1 antibody) variant comprises amino acid substitutions at positions S298A, E333A, and K334A of the Fc region.

[0309] In some embodiments, alterations in the Fc region result in altered (ie, enhanced or reduced) C1q binding and / or complement-dependent cytotoxicity (CDC), as described in US Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0310] In some embodiments, an anti-O1 antibody (e.g., a full-length anti-O1 antibody) variant is provided, comprising an Fc region variant having one or more amino acid substitutions that extend half-life or enhance binding to an Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US 2005 / 0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance FcRn binding. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( U.S. Pat. No. 7,371,826 ).

[0311] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc region variants.

[0312] The present application contemplates anti-O1 antibodies (eg, full-length anti-O1 antibodies) comprising any one of the Fc variants described herein, or a combination thereof.

[0313] Glycosylation variants

[0314] In some embodiments, the anti-O1 antibodies (e.g., full-length anti-O1 antibodies) provided herein are altered to increase or decrease the extent of glycosylation of the anti-NGF antibody. Adding or deleting glycosylation sites on the anti-O1 antibody can be conveniently accomplished by altering the amino acid sequence of the anti-NGF antibody or a polypeptide portion thereof to add or remove one or more glycosylation sites.

[0315] Wherein the anti-O1 antibody comprises an Fc region, the sugars attached thereto can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are typically N-linked to the CH2 domain of the Fc region at Asn297 (see, for example, Wright et al., TIBTECH 15:26-32 (1997)). Such oligosaccharides can include 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. In some embodiments, the anti-O1 antibodies of the present application can be subjected to oligosaccharide modification to generate anti-O1 antibody variants with certain improved properties.

[0316] The N-glycans attached 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). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for binding to FcγRs; however, non-fucosylated N-glycans enhance the binding of Fc to FcγRIIIa. Enhanced binding to FcRIIIa results in enhanced ADCC, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0317] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or CH2 domain is non-glycosylated. In some embodiments, glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.

[0318] In some embodiments, anti-O1 antibody variants (e.g., full-length anti-O1 antibodies) are provided that comprise an Fc region in which the carbohydrate structures attached to the Fc region have reduced or absent fucose, which may enhance ADCC function. Specifically, anti-O1 antibodies are provided herein that have reduced fucose relative to the same anti-O1 antibody produced in wild-type CHO cells. That is, they are characterized by having a lower amount of fucose compared to antibodies produced in native CHO cells (e.g., CHO cells that produce native glycosylated forms, CHO cells that contain the native FUT8 gene). In some embodiments, the N-linked glycans of the anti-O1 antibody have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the anti-O1 antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycans of the anti-O1 antibody do not contain fucose, i.e., the anti-O1 antibody is completely fucose-free, has no fucose, or is defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all glycostructures (e.g., complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.), US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include 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). Cell lines capable of producing defucosylated antibodies include Lec13 CHO 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 CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO 2003 / 085107).

[0319] Anti-O1 antibody (e.g., full-length anti-O1 antibody) variants further provide bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the anti-O1 antibody is bisected by GlcNAc. Such anti-O1 antibody (e.g., full-length anti-O1 antibody) variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Anti-O1 antibody (e.g., full-length anti-O1 antibody) variants are also provided that have at least one galactose residue in the oligosaccharide attached 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.).

[0320] In some embodiments, the anti-O1 antibody (e.g., full-length anti-O1 antibody) variant comprises an Fc region that binds to FcγRIII. In some embodiments, 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 has enhanced ADCC activity in the presence of human effector cells compared to an otherwise identical anti-O1 antibody (e.g., full-length anti-O1 antibody) having a human wild-type IgG1 Fc region.

[0321] Cysteine engineered variants

[0322] In some embodiments, it is desirable to prepare cysteine-engineered anti-O1 antibodies (e.g., full-length anti-O1 antibodies) in which one or more amino acid residues are substituted with cysteine residues. In some embodiments, the substituted residues occur at accessible sites of the anti-O1 antibody. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the anti-O1 antibody, which can be used to conjugate the anti-O1 antibody to other moieties, such as drug moieties or linker-drug moieties, to prepare anti-K. pneumoniae O1 antigen immunoconjugates as further described herein. Cysteine-engineered anti-O1 antibodies (e.g., full-length anti-O1 antibodies) can be prepared, for example, as described in U.S. Pat. No. 7,521,541.

[0323] derivative

[0324] In some embodiments, the anti-O1 antibodies (e.g., full-length anti-O1 antibodies) provided herein can be further modified to include other non-protein moieties known in the art and readily available. Suitable moieties 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, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have 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. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, whether it is desired to improve the properties or function of the anti-O1 antibody, whether the anti-O1 antibody derivative is intended for treatment of a particular condition, etc.

[0325] Pharmaceutical composition

[0326] Also provided herein are compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) 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. In some embodiments, a pharmaceutical composition is provided comprising any of the anti-O1 antibodies described herein and a pharmaceutically acceptable carrier.

[0327] Suitable anti-O1 antibody formulations can be obtained by mixing an anti-O1 antibody having the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and prepared in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphates, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418, expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration, and the reconstituted formulation can be administered subcutaneously to the subject to be treated herein. Cationic liposomes or liposomes can be used to deliver the anti-O1 antibodies of the present application to cells.

[0328] In addition to the anti-O1 antibody (e.g., full-length anti-O1 antibody), the formulations described herein may also contain one or more other active substances necessary to treat a specific condition, preferably substances with complementary activities and no adverse reactions with each other. For example, in addition to the anti-O1 antibody, it may be desirable to further include antibiotics, steroids, non-steroidal inflammatory suppressants, and other antibacterial and anti-inflammatory agents. These molecules are present in combination in amounts effective for their intended purpose. The effective amount of the other substances depends on the amount of anti-O1 antibody in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are generally used in the same dosages and routes of administration as described herein, or at 1% to 99% of currently used dosages.

[0329] The anti-O1 antibody (e.g., full-length anti-O1 antibody) can also be encapsulated in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Sustained-release formulations can be prepared.

[0330] Sustained-release formulations of anti-O1 antibodies (e.g., full-length 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), which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for over 100 days, certain hydrogels can release proteins over shorter time periods. When encapsulated antibodies remain in the body for extended periods, they can denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of bioactivity or altered immunogenicity. Rational strategies can be designed to stabilize anti-O1 antibodies based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be intermolecular SS bonds formed through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0331] In some embodiments, the anti-O1 antibody (eg, full-length anti-O1 antibody) is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.

[0332] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0333] Treatment or prevention using anti-O1 antibodies

[0334] Anti-O1 antibodies (e.g., full-length anti-O1 antibodies) and / or compositions described herein can be administered to an individual (e.g., a mammal, such as a human) to treat or prevent diseases and / or conditions associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetifera, and / or Klebsiella rhinosclerosis) (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections, and infections associated with Klebsiella (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, and other diseases associated with infections caused by Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis), including but not limited to pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. Therefore, in some embodiments, the present application provides a method for treating or preventing diseases and / or conditions associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetoriosa, and / or Klebsiella rhinosclerosis) (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with Klebsiella (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetoriosa, and / or Klebsiella rhinosclerosis) infection), comprising administering to a subject an effective amount of a composition (e.g., a pharmaceutical composition) comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), such as any of the anti-O1 antibodies (e.g., full-length anti-O1 antibodies) described herein. In some embodiments, the subject is human.

[0335] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably a Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetoriosa, and / or Klebsiella rhinosclerosis) is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-01 antibody (e.g., a full-length anti-01 antibody) that specifically binds to a Klebsiella pneumoniae 01 antigen. In some embodiments, the anti-01 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0336] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably serotype Klebsiella 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the anti-O1 antibody comprises: 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V Lcomprising: 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 the V L A variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0337] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V H 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 V L , the V L The invention relates to a polypeptide comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 46.

[0338] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0339] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO: 19, LC-CDR2, which comprises the amino acid sequence of SEQ ID NO: 26, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 33, or the V LA variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0340] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V H 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 V L , the V L The invention also comprises an amino acid sequence of SEQ ID NO: 47 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 47.

[0341] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0342] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0343] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V Hcomprising 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 V L , the V L The invention also comprises an amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 48.

[0344] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0345] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V LA variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0346] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V H comprising the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 42; and V L , the V L The invention also comprises an amino acid sequence of SEQ ID NO: 49 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 49.

[0347] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0348] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0349] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V Hcomprising 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 V L , the V L The invention also comprises an amino acid sequence of SEQ ID NO: 50 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 50.

[0350] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0351] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; 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, which comprises the amino acid sequence of SEQ ID NO: 37, or the V LA variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0352] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V H 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 V L , the V L The invention also provides a method for preparing a polypeptide comprising the amino acid sequence of SEQ ID NO: 51 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 51.

[0353] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0354] For example, in some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody), wherein the antibody comprises: V H , the V H 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 the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , which 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, or the V L A variant of the present invention comprising up to about 5 amino acid substitutions in its LC-CDRs. In some embodiments, the anti-O1 antibody is a full-length antibody. In some embodiments, the full-length anti-O1 antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from, for example, pneumonia, urinary tract infection, sepsis / bacteremia / sepsis, neonatal sepsis / bacteremia / sepsis, diarrhea, soft tissue infection, post-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathy. In some embodiments, the individual is human.

[0355] In some embodiments, a method for treating or preventing a disease associated with infection or colonization with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine, Klebsiella granulomatosa, Klebsiella fetida, and / or Klebsiella rhinosclerosis) in an individual is provided, comprising administering to the individual an effective amount of a composition comprising an anti-O1 antibody, wherein the antibody comprises: V H , the V Hcomprising 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 V L , the V L The invention also comprises an amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant has at least about 90% sequence identity with the amino acid sequence of SEQ ID NO: 52.

[0356] In some embodiments, the anti-O1 antibodies described herein are full-length anti-O1 antibodies comprising an IgG1 or IgG4 constant region. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 53. In some embodiments, the heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 54. In some embodiments, 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.

[0357] In some embodiments, any of the methods of treatment or prevention described herein further provides a therapeutic or preventive effect for diseases and / or conditions associated with Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetorhinis, and / or Klebsiella rhinosclerosis). In some embodiments, the method can prevent Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetorhinis, and / or Klebsiella rhinosclerosis) infection.

[0358] In some embodiments, the subject is a mammal (e.g., a human, non-human primate, rat, mouse, cattle, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the subject is a human. In some embodiments, the subject is a clinical patient, a clinical trial volunteer, an experimental animal, etc. In some embodiments, the subject 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 subject is greater than 60 years old (including, for example, greater than 70, 80, 90, or 100 years old). In some embodiments, the individual is a subject or a contact of a subject diagnosed with Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetifera and / or Klebsiella rhinosclerosis) infection or is immunocompromised or immunosuppressed so as to be susceptible to Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetifera and / or Klebsiella rhinosclerosis) infection (e.g., nosocomial infection, opportunistic infection, post-organ transplant infection, and other diseases associated with Klebsiella infection).

[0359] In some embodiments, the individual has one or more risk factors associated with infection with Klebsiella, preferably Klebsiella serotype 01 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithine-lyticus, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis). For example, in some embodiments, the individual has exposed or disrupted the mucus layer of the skin. In some embodiments, the individual has one or more burns. In some embodiments, the individual has one or more surgical wounds. In some embodiments, the individual has a skin disease. In some embodiments, the individual has a foreign body implanted, such as, but not limited to, a respirator or catheter. In some embodiments, the individual has been diagnosed with or is genetically susceptible to an immunodeficiency disease, including but not limited to HIV infection, AIDS, and / or neutropenia. In some embodiments, the individual has received one or more forms of chemotherapy. In some embodiments, the individual has received one or more forms of adrenal glucocorticoid therapy. In some embodiments, the individual has received one or more forms of chemotherapy. In some embodiments, the individual is diagnosed with or is genetically predisposed to cancer, diabetes, and / or chronic structural lung disease (e.g., cystic fibrosis or COPD). In some embodiments, the individual is diagnosed with or is genetically predisposed to dysbiosis of the digestive system and / or other organs. In some embodiments, the individual has one or more risk factors associated with one or more of the above diseases or conditions.

[0360] Numerous diagnostic methods and clinical descriptions of nosocomial infections, opportunistic infections, post-transplant infections, and other diseases associated with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella fetifera, and / or Klebsiella rhinosclerosis) are known in the art. Such methods include, but are not limited to, immunohistochemistry, ELISA, PCR, Western blotting, and fluorescence in situ hybridization (FISH).

[0361] In some embodiments, the anti-O1 antibodies (e.g., full-length anti-O1 antibodies) and / or compositions described herein are used in combination with a second, third, or fourth agent (e.g., antibiotics, steroids and non-steroidal inflammatory suppressants, and / or other antibacterial or anti-inflammatory agents) to treat or prevent diseases associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinelyticus, Klebsiella granulomatis, Klebsiella febrifuge, and / or Klebsiella rhinosclerosis).

[0362] In some embodiments, the antibiotic is a penicillin, a cephalosporin, a carbapenem, a fluoroquinolone, an aminoglycoside, a monoamine, a polymyxin, an antibiotic composition containing a beta-lactamase inhibitor, or any combination thereof. In some embodiments, the antibiotic is cefepime, ceftazidime, cefpirome, imipenem, meropenem, ticarcillin, piperacillin, azlocillin, carbenicillin, mezlocillin, atrenam, tobramycin, gentamicin, amikacin, ciprofloxacin, levofloxacin, cefoperazone sulbactam, piperacillin-tazobactam, fosfomycin, or any combination thereof. In some embodiments, the antibiotic is one or more of imipenem, tobramycin, ciprofloxacin, meropenem, or atrenam. In some embodiments, the antibiotic is one or more of gentamicin, ampicillin, or kanamycin.

[0363] Dosage and method of administration of anti-O1 antibodies

[0364] The dosage of an anti-O1 antibody (e.g., isolated anti-O1 antibody) composition administered to an individual (e.g., a human) may vary depending on the specific composition, the mode of administration, and the type of disease being treated. In some embodiments, the amount of the composition (e.g., a composition comprising an anti-O1 antibody) is effective to produce an objective response (e.g., a partial response or a complete response) in the treatment of nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella planticola, Klebsiella terrestris, Klebsiella ornithinelyticus, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis). In some embodiments, the amount of the anti-O1 antibody composition is sufficient to produce a complete response in the individual. In some embodiments, the amount of the anti-O1 antibody composition is sufficient to produce a partial response in the individual. In some embodiments, the dose of the anti-O1 antibody composition administered (e.g., when administered alone) is sufficient to produce an overall response rate greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% in a population of individuals treated with the anti-O1 antibody composition. A subject's response to treatment methods described herein can be determined, for example, by detection of Klebsiella using Gram staining or other phenotypic testing methods.

[0365] In some embodiments, the amount of the composition (e.g., a composition comprising an isolated anti-O1 antibody) is sufficient to prolong progression-free survival in an individual. In some embodiments, the amount of the composition is sufficient to prolong overall survival in an individual. In some embodiments, the amount of the composition (e.g., when administered alone) is sufficient to produce a clinical benefit greater than 50%, 60%, 70%, or 77% in a population of individuals treated with the anti-O1 antibody composition.

[0366] In some embodiments, the amount of the composition (e.g., a composition comprising an isolated anti-O1 antibody), alone or in combination with a second, third, and / or fourth agent, is sufficient to reduce the magnitude of Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae) organ burden by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the same subject before treatment or compared to the corresponding organ burden in other untreated subjects. The magnitude of this therapeutic effect can be measured using standard methods, such as in vitro assays with purified enzymes, cell-based assays, animal models, or human studies.

[0367] In some embodiments, the amount of anti-O1 antibody (e.g., full-length anti-O1 antibody) in the composition is below a level that causes a toxic effect (i.e., an effect above a clinically acceptable toxicity level) when the composition is administered to a subject, or is at a level at which potential side effects are manageable or tolerable.

[0368] In some embodiments, following the same dosing regimen, the amount of the composition is close to 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.

[0369] In some embodiments, the amount of anti-O1 antibody (eg, full-length anti-O1 antibody) in the composition is in the range of 0.001 μg to 1000 μg.

[0370] In any of the above embodiments, the effective amount of the anti-O1 antibody (eg, full-length anti-O1 antibody) in the composition is in the range of 0.1 μg / kg to 100 mg / kg, calculated based on body weight.

[0371] The anti-O1 antibody composition can be administered to an individual (e.g., a human) by a variety of routes, including, for example, intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhaled, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, mucosal, or transdermal administration. In some embodiments, a sustained-release formulation of the composition is used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered intraarterially. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered by hepatic artery infusion. In some embodiments, the composition is administered to a site distal to the primary lesion.

[0372] Products and kits

[0373] In some embodiments of the present application, an article of manufacture is provided, comprising a substance for treating or preventing Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis) infection in an individual, or for delivering an anti-O1 antibody (e.g., a full-length anti-O1 antibody) to cells attached to LPS-expressing pathogens. The article of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition effective for treating or preventing a disease or condition described herein and has a sterile port (e.g., the container can be an intravenous infusion bag or a vial with a cap pierceable by a hypodermic needle). The at least one active substance in the composition is the anti-O1 antibody described herein. The label or package insert indicates that the composition can be used to treat or prevent a specific condition. The label or package insert further includes instructions for administering the anti-O1 antibody composition to a patient. Articles of manufacture and kits comprising combination therapies are also contemplated herein.

[0374] Package insert refers to instructions typically included in commercial packaging of therapeutic or prophylactic products, which contain information about indications, usage, dosage, administration, contraindications and / or warnings related to the use of these therapeutic or prophylactic products. In some embodiments, the package insert indicates that the composition can be used to treat or prevent diseases associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinelyticus, Klebsiella granulomatis, Klebsiella febrileus, and / or Klebsiella rhinosclerosis)). In some embodiments, 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-organ transplant infection, surgical infection, wound infection, lung infection, pyogenic liver abscess (PLA), lung abscess, cellulitis, necrotizing fasciitis, myositis, endophthalmitis, peritonitis, meningitis, necrotizing meningitis, ankylosing spondylitis or spondyloarthropathy.

[0375] In addition, 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, Ringer's solution, or dextrose solution. Other materials desirable from a commercial and user perspective may also be included, including other buffers, diluents, filters, needles, and syringes.

[0376] Also provided are kits that can be used for various purposes, such as for treating or preventing diseases associated with Klebsiella serotype O1 infection (e.g., nosocomial infections, opportunistic infections, post-transplant infections, and other diseases associated with Klebsiella infection), preferably Klebsiella pneumoniae, preferably Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithines, Klebsiella granulomatosa, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis), or for delivering anti-O1 antibodies (e.g., full-length anti-O1 antibodies) to cells attached to pathogens expressing LPS, optionally in combination with a product. The kits of the present application include one or more containers containing an anti-O1 antibody composition (or a single-dose form and / or product), and in some embodiments, further include another agent (e.g., an agent described herein) and / or instructions for use in accordance with any of the methods described herein. The kits may further include instructions for selecting a suitable subject for treatment or prevention. The instructions for use included in the kit of this application are usually written instructions on a label or package insert (such as a paper sheet included in the kit), and machine-readable instructions (such as instructions on a magnetic or optical storage disc) are also acceptable.

[0377] For example, in some embodiments, the kit includes a composition comprising an anti-O1 antibody (e.g., a full-length anti-O1 antibody). In some embodiments, the kit includes: a) a composition comprising any of the anti-O1 antibodies described herein, and b) at least one other agent in an effective amount that enhances the efficacy (e.g., therapeutic efficacy, detection efficacy) of the anti-O1 antibody. In some embodiments, the kit includes: a) a composition comprising any of the anti-O1 antibodies described herein, and b) instructions for administering the anti-O1 antibody composition to a subject for treating or preventing a disease associated with infection with a Klebsiella serotype, preferably a Klebsiella serotype O1 (e.g., Klebsiella pneumoniae, Klebsiella oxytoca, Klebsiella plantarum, Klebsiella terrestris, Klebsiella ornithinelyticus, Klebsiella granulomatis, Klebsiella febrifolium, and / or Klebsiella rhinosclerosis) (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with Klebsiella infections). In some embodiments, the kit includes: a) a composition comprising any of the anti-O1 antibodies described herein, and b) at least one other agent in an effective amount that enhances the efficacy (e.g., therapeutic efficacy, detection efficacy) of the anti-O1 antibody, and c) instructions for administering the anti-O1 antibody composition and other agent to a subject for treating or preventing a disease associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., nosocomial infections, opportunistic infections, post-organ transplant infections, and other diseases associated with Klebsiella infection). The anti-O1 antibody and other agent can be present in separate containers or in the same container. For example, the kit can include one specific composition or two or more compositions, wherein one composition includes the anti-O1 antibody and another composition includes another agent.

[0378] In some embodiments, the kit comprises a nucleic acid (or a set of nucleic acids) encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody), and b) a host cell expressing the nucleic acid (or nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody), and b) instructions for: i) expressing the anti-O1 antibody in the host cell, ii) preparing a composition comprising the anti-O1 antibody, and iii) administering the composition comprising the anti-O1 antibody to a subject to treat or prevent a disease associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., nosocomial infection, opportunistic infection, post-organ transplant infection, and other diseases associated with Klebsiella infection). In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an anti-O1 antibody (e.g., a full-length anti-O1 antibody), b) a host cell expressing the nucleic acid (or a set of nucleic acids), and c) instructions for: i) expressing the anti-O1 antibody in the host cell, ii) preparing a composition comprising the anti-O1 antibody, and iii) administering the composition comprising the anti-O1 antibody to an individual to treat or prevent a disease associated with infection with Klebsiella, preferably Klebsiella serotype O1 (e.g., nosocomial infection, opportunistic infection, post-organ transplant infection, and other diseases associated with Klebsiella infection).

[0379] The test kits described herein are packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The test kits may optionally provide other components, such as buffers and instructional information. Therefore, the application also provides articles, which include vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc.

[0380] Instructions for use of the anti-O1 antibody composition typically include information such as dosage, dosing cycle, and route of administration. The container can be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. For example, a kit is provided that contains a sufficient dose of an anti-O1 antibody (e.g., a full-length anti-O1 antibody) as described herein to provide long-term, effective treatment for an individual, e.g., one week, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, three weeks, four weeks, six weeks, eight weeks, three months, four months, five months, seven months, eight months, nine months, or longer. The kit can also contain multiple unit doses of the anti-O1 antibody, the pharmaceutical composition, and instructions for use, and is packaged in an amount sufficient for storage and use in a pharmacy, e.g., a hospital pharmacy or compounding pharmacy.

[0381] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way. DETAILED DESCRIPTION

[0382] In the following disclosed examples, KP19173 represents Klebsiella pneumoniae serotype O1 whose LPS contains two types of repeating units, D-gal I and D-gal II ( Figure 1A ); KP19213 represents the Klebsiella pneumoniae O1 serotype whose LPS contains two types of repeating units, D-gal Ⅲ and D-gal Ⅱ ( Figure 1B ); KP19180 represents Klebsiella pneumoniae serotype O2 whose LPS contains D-gal Ⅰ repeating units ( Figure 1C ); KP19203 represents Klebsiella pneumoniae serotype O2 whose LPS contains D-gal Ⅲ repeating units ( Figure 1D In the embodiments of the present invention, the anti-O2 antibody KPN70 (binding to D-gal Ⅰ repeating unit) disclosed in patent application CN109689090A, the anti-O antigen antibody G3-78 (binding to D-gal Ⅲ repeating unit) disclosed in patent application CN107371365A, and / or the anti-O1 antibody MPG196 (binding to D-gal II repeating unit) disclosed in patent application WO2018 / 029356A1 were used as control antibodies.

[0383] Example 1: Preparation of LPS and O-PS and Screening of Single-chain Fv Antibodies (scFv) Against O1

[0384] Preparation of LPS

[0385] Pick a single clone of Klebsiella pneumoniae (such as KP19173, KP19213, KP19180 or KP19203, etc.) and inoculate it into 2×YT medium for expansion culture. Collect the bacteria by centrifugation at 12000rpm and 4℃, and discard the supernatant. After resuspending the bacterial precipitate in sterile water, place it in a -80℃ refrigerator and freeze and thaw it repeatedly three times to break the bacteria. Use the hot phenol water method to extract LPS: first, add 555mL of 90% phenol solution to 600mL of bacterial suspension, stir continuously in a 65℃ water bath, and remove it after 1h. After cooling to 4℃ in a cold water bath, centrifuge at 20000g and 4℃ for 30min. After centrifugation, the solution is divided into 3 layers, from top to bottom, namely, water layer, phenol layer and insoluble layer. The upper aqueous phase contains LPS, the middle layer is denatured protein, and the lower layer is the precipitate. Carefully pipette the upper aqueous phase into a new tube, and add 6 times the volume of 95% ethanol, and at the same time add sodium acetate with a final concentration of 0.1%. After the mixture is ice-bathed for 10 minutes, white dot-like precipitates are clearly observed. Centrifuge at 20,000g and 4°C for 1 hour, and discard the supernatant. Completely resuspend the precipitate with 70% ethanol, centrifuge at 13,000rpm and 4°C for 15 minutes, and discard the supernatant. Place the centrifuge tube in a clean bench and blow dry to obtain the crude LPS extract. Prepare the crude extract into a 2% (w / v) solution, add an equal amount of 95% ethanol, and stir while adding. Centrifuge at 12,000rpm for 30 minutes, and remove the precipitate (repeat twice). Take the supernatant and place it in a new tube, and add 6 times the volume of 95% ethanol, and at the same time add sodium acetate with a final concentration of 0.1%. Ice-bathed for 10 minutes, centrifuged at 20,000g and 4°C for 30 minutes, and discard the supernatant. Resuspend the precipitate in 100 mL of 70% ethanol, centrifuge at 20,000 g, 4°C for 30 min, discard the supernatant, and vacuum dry the precipitate for later use.

[0386] Preparation of O-PS

[0387] Weigh 1 g of LPS prepared in the above process and add it to 200 mL of 1% acetic acid solution. Incubate in a boiling water bath for 45 minutes. After cooling, ultracentrifuge at 105,000 g at 4°C for 2 hours. The precipitate is lipid A, and the supernatant is O-PS. Remove the supernatant and concentrate the O-PS by vacuum centrifugation; then dissolve in water for later use.

[0388] Preparation of biotinylated O-PS

[0389] Add an appropriate amount of O-PS solution to a 3 kDa ultrafiltration tube and centrifuge at 4000 rpm for 20-30 min. Add 0.2 M boric acid buffer (pH 9.0) to the ultrafiltration tube and centrifuge at 4000 rpm. Repeat this step three times. Dissolve the O-PS in 0.2 M boric acid buffer (pH 9.0). Determine the polysaccharide concentration in the O-PS solution using the sulfuric acid-phenol colorimetric method. Calculate the total amount of polysaccharide based on the solution volume (Zhang Qing, Zhang Tianmin. Determination of polysaccharide content by phenol-sulfuric acid colorimetry [C]. / / Proceedings of the National Conference on Biochemical New Drug Research and Clinical Application of the Chinese Pharmaceutical Association. Qingdao: Biochemistry and Biotechnology Drug Professional Committee of the Chinese Pharmaceutical Association, 2004: 176-178.). An appropriate amount of Amine-PEG11-Biotin (Thermo, 26136) was added to the O-PS solution at a mass ratio of 1.35:1. The solution was incubated at 37°C for 4 days. At 1, 24, 48, and 72 hours, 180 μL of ALD Coupling Buffer (Sterogene, 9704) was added per mL of the reaction solution. The solvent of the biotinylated O-PS was replaced with PBS using a 3 kDa ultrafiltration tube. The resulting solution was the biotinylated O-PS (designated Bio-OPS).

[0390] Screening of single-chain Fv (scFv) antibodies against O1 antigen

[0391] Construction of scFv antibody yeast display library: RNA was extracted from 2000 human blood samples and cDNA was obtained by reverse transcription. H and V K Specific primers amplify V H and V K The fragment was purified by gel recovery and ligated to V H and V K scFv was constructed and cloned into the yeast display plasmid PYD1, which was then electroporated into yeast to obtain a scFv antibody yeast display library.

[0392] Screening for single-chain Fvs (scFvs) against the O1 antigen: After several rounds of panning, scFvs that bind to K. pneumoniae O-PS were isolated from the yeast display library. Briefly, yeast cells that bind to K. pneumoniae O-PS were enriched using MACS magnetic bead sorting. Yeast cells at 1000 OD were centrifuged at 2500g for 5 minutes. The resulting cell pellet was resuspended in 1 L of SGCAA medium at a starting concentration of OD600 = 1 and induced for expression at 20°C and 250 rpm for 40-48 hours. The cell culture was centrifuged and washed with PBSM solution. The cell pellet was resuspended in 5-10 volumes of PBSM solution containing 1 μM Bio-OPS and incubated at 4°C for 1 hour. After centrifugation and PBSM washing, unbound antigen was washed away with PBSM solution. After adding magnetic beads, the mixture was thoroughly mixed and then incubated on a 4°C rotating machine for 30 minutes. Centrifuge at 2500g for 5 minutes, discard the supernatant, and resuspend the pellet in 5-10 volumes of PBSM. Add 7 mL of cell suspension to the column at a time until all the cell suspension has flowed through the column. Elute and collect cells bound to the column for culture and subsequent FACS sorting.

[0393] FACS screening of single-chain Fv antibodies (scFvs) against the O1 antigen: Yeast enriched after MACS panning were subjected to flow cytometry sorting (FACS). Briefly, yeast cells induced in SGCAA medium were pelleted, washed with 1 mL of PBSM, and centrifuged at 14,000 g for 30 seconds. The yeast cells were resuspended in 100 μL of PBSM buffer containing Bio-OPS and incubated at room temperature for 1 hour. After washing, the cells were stained with SA-PE (BD, 554061) and anti-V5 antibody (GenScript, A01803) in 100 μL of PBSM buffer and incubated on ice for 20 minutes. The top 1% of double-positive cells were selected and sorted into culture medium for cell expansion. Bio-OPS-guided selection using the KP19173 strain and / or Bio-OPS-guided selection using the KP19213 strain were repeated for 2-3 cycles, respectively. Individual clones were further analyzed by FACS analysis. After the screening process, a series of positive scFv antibodies were obtained and sequenced.

[0394] Example 2: Preparation and characterization of full-length anti-O1 antibodies

[0395] Preparation of full-length anti-O1 antibody

[0396] The obtained positive scFv antibodies were reconstructed into human IgG1 or IgG4 full-length antibody molecules containing the heavy chain constant region of human IgG1 or IgG4 and the human kappa light chain constant region. V was amplified from the yeast expression vector. L and VH , respectively constructed into the eukaryotic expression vectors pTT5-L (containing the kappa constant region) and pTT5-H1 (containing the IgG1 heavy chain constant region) or pTT5-H4 (containing the 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% CO2, and 120 rpm for 5 days, and the culture fluid was purified using a Protein A affinity chromatography column. Briefly, the Protein A column was first equilibrated with 6 column volumes of 50 mM PBS buffer (containing 0.15 M NaCl, pH 7.2) at a flow rate of 150 cm / h. The culture supernatant (adjusted to pH 7.2) was passed through the column at a flow rate of 150 cm / h. After further equilibration of the column, elution was performed with 50 mM sodium citrate buffer (pH 3.5), and the eluate was collected. The obtained full-length antibody was subjected to further biochemical and biological activity analysis.

[0397] ELISA binding assay

[0398] The prepared full-length anti-O1 monoclonal antibody (reconstructed adult IgG1 form) was subjected to a binding test with LPS prepared in Example 1 (obtained from KP19173 strain or KP19213 strain, respectively). The test was used to identify antibodies that bind to Klebsiella pneumoniae LPS, while KPN70, G3-78, and MPG196 were set as control antibodies. Briefly, LPS was dissolved in PBS solution (adjusted to pH 7.2, final concentration of 0.2 μg / mL), coated on a 96-well plate at 100 μL / well, and incubated at 4°C overnight. Before adding the antibody, the 96-well plate was washed 5 times with 200 μL / well PBST solution. 200 μL of 10% BSA (Beyotime Biotechnology, ST023-200g) was added to each well and incubated at 37°C for 1 hour. Washed 5 times with PBST solution. Each antibody sample was first diluted to 1 μg / mL, followed by a gradient dilution at a ratio of 1:3. The gradient diluted antibody samples were added to a 96-well plate, 50 μL per well, and incubated at 37°C for 1 hour. Then washed 5 times with PBST solution. 100 μL of secondary antibody (goat anti-human IgG-HRP (Beyotime Biotechnology, A0201) (1:10000)) was added to each well and incubated at 37°C for 1 hour. Washed 5 times with PBST solution. 100 μL of TMB (southern biotech, 0410-01) was added to each well and incubated at 37°C for 10-20 minutes. The reaction was terminated with 2M H2SO4, OD450 was read, and the binding curve was generated by Graphpad Prism to calculate EC 50 value.

[0399] The results in Table 5 show that the anti-O1 antibodies G1-G7 and the control antibody MPG196 bind to both KP19173 and KP19213 LPS. Compared to the control antibody MPG196, the anti-O1 antibodies G1-G7 bind to both KP19173 and KP19213 LPS at a comparable or superior capacity. In contrast, the control antibody KPN70 binds only to KP19173 LPS, and the control antibodies G3-78 bind only to KP19213 LPS.

[0400] Table 5

[0401]

[0402]

[0403] LPS neutralization assay

[0404] Commercial reporter cell line HEK-Blue TM hTLR4 (Invivogen, hkb-htlr4) stably expresses human TLR4, MD-2, and CD14 co-receptors, as well as NF-κB-induced secreted embryonic alkaline phosphatase (SEAP). Bacterial LPS can trigger Toll-like receptor 4 (TLR-4) signaling in this cell line, leading to the activation of downstream NF-κB transcription factors and the secretion of SEAP, which can be expressed by QUANTI-Blue TM Briefly, HEK-Blue was cultured according to the manufacturer's instructions. TM hTLR4 cells were seeded into 96-well culture plates, with approximately 2.5 × 10 cells per well. 4 cells; then, 10 μL of LPS (10 μg / mL, derived from KP19173 strain or KP19213 strain) prepared in Example 1 and a gradient dilution of anti-O1 antibody (reconstituted adult IgG1 form) with a starting concentration of 1 mg / mL were pre-mixed and added to a 96-well culture plate and incubated at 37°C, 5% CO2 for 6-16 hours. Then, 40 μL of supernatant was mixed with 160 μL of preheated QUANTI-Blue TM The cells were mixed with the anti-O1 antibody (Invivogen) solution and incubated for 60-90 minutes. The absorbance was read at 620-655 nm. KPN70, G3-78, and MPG196 were also set as control antibodies. The inhibition rates under LPS stimulation alone and without LPS stimulation were calculated as 0% and 100%, res...

Claims

1. An isolated antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O1 antigen, comprising: V H , comprising: HC-CDR1, whose amino acid sequence is SEQ ID NO: 2, HC-CDR2, whose amino acid sequence is SEQ ID NO: 8, and HC-CDR3, whose amino acid sequence is SEQ ID NO: 14; and V L , which comprises: LC-CDR1, whose amino acid sequence is SEQ ID NO: 19, LC-CDR2, whose amino acid sequence is SEQ ID NO: 26, and LC-CDR3, whose amino acid sequence is SEQ ID NO:

33.

2. The isolated antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O1 antigen according to claim 1, comprising: V H , whose amino acid sequence is SEQ ID NO: 40 or a variant thereof, said variant having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 40; and V L , whose amino acid sequence is SEQ ID NO: 47 or a variant thereof, wherein the variant has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

47.

3. The isolated antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen according to claim 1 or 2, wherein the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen comprises an Fc fragment.

4. The isolated antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen according to claim 3, wherein the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen is a full-length IgG antibody.

5. The isolated antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen according to claim 4, wherein the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen is a full-length IgG1, IgG2, IgG3 or IgG4 antibody.

6. The isolated antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen according to claim 1 or 2, wherein the antibody or antigen-binding fragment that specifically binds to Klebsiella pneumoniae O1 antigen is a chimeric, humanized or fully human antibody.

7. The isolated antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen according to claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody, Fv fragment, diabody and linear antibody.

8. A nucleic acid molecule encoding the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen according to any one of claims 1 to 7.

9. An isolated vector comprising the nucleic acid molecule of claim 8.

10. An isolated host cell comprising the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, or the vector according to claim 9.

11. A method for preparing an antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O1 antigen, comprising: a) culturing the host cell of claim 10 under conditions effective to express an antibody or antigen-binding fragment that specifically binds to a Klebsiella pneumoniae O1 antigen; and b) obtaining the expressed antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen from the host cell.

12. A pharmaceutical composition comprising the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9, the isolated host cell of claim 10, or the antibody or antigen-binding fragment prepared by the method of claim 11, and a pharmaceutically acceptable carrier.

13. Use of the antibody or antigen-binding fragment that specifically binds to the Klebsiella pneumoniae O1 antigen of any one of claims 1 to 7, the nucleic acid molecule of claim 8, the vector of claim 9, the isolated host cell of claim 10, the antibody or antigen-binding fragment produced by the method of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating, preventing, or ameliorating a disease or condition in an individual of interest, wherein the disease or condition is associated with infection or colonization with Klebsiella pneumoniae.

14. The use according to claim 13, wherein the disease or disorder is a lung infection.

15. The use according to claim 14, wherein the lung infection comprises pneumonia and lung abscess.

16. The use according to claim 13, wherein the disease or condition is urinary tract infection.

17. The use according to claim 13, wherein the disease or disorder is diarrhea.

18. The use according to claim 13, wherein the disease or disorder is a soft tissue infection.

19. The use according to claim 18, wherein the soft tissue infection comprises cellulitis.

20. The use according to claim 13, wherein the disease or condition is wound infection.

21. The use according to claim 20, wherein the wound infection comprises a surgical infection.

22. The use according to claim 21, wherein the surgical infection comprises an infection after organ transplantation.

23. The use according to claim 13, wherein the disease or condition is selected from the group consisting of pyogenic liver abscess, necrotizing fasciitis, myositis, endophthalmitis, meningitis.

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