Anti-O2 antibodies and uses thereof

BACKGROUND OF THE INVENTION
Field of the Invention

The field of the invention generally relates to antigen binding proteins (e.g., antibodies and antigen-binding fragments thereof) that specifically bind to Klebsiella pneumoniae O2 antigen and the use of those binding proteins for prevention or treatment of Klebsiella infections.

Background of the Invention

Klebsiella is a Gram negative bacterium that is rapidly gaining clinical importance as a causative agent for opportunistic and nosocomial infection, including pneumonia, urinary tract infection, neonatal septicemia, and surgery wound infection. In addition, there are emerging syndromes associated with Klebsiella infections such as pyogenic liver abscesses (PLA), endophthalmitis, meningitis, and necrotizing meningitis. (See Iredell et al. BMJ 351: h6420 (2015).)

Antibiotic resistance has emerged as one of the major challenges in the fight against bacterial infections. While some progress has been made against drug resistant Staphylococcus aureus, Gram negative opportunistic infections are most problematic. Among these, Klebsiella pneumoniae has become particularly challenging with multi-drug resistant strains widely circulating. Antibiotic resistances such as Extended-Spectrum Beta Lactamase (ESBL), K. pneumoniae carbapenemase (KPC), and New Delhi metallo-beta-lactamase 1 (NDM-1) have spread worldwide and rendered current antibiotic classes largely inadequate. This reality coupled with the dwindling antibiotics pipeline leaves few therapeutic alternatives. Several recent high profile outbreaks underscore the urgency associated with K. pneumoniae antibiotic resistance. It is therefore critical to develop strategies to complement antibiotics therapies.

Multiple virulence factors have been implicated in K. pneumoniae pathogenesis, including capsular polysaccharides (CPS) and lipopolysaccharides (LPS). Polyclonal antibodies directed against LPS and CPS are protective in preclinical models of lethal K. pneumoniae infections. However targeting these two antigens with antibodies poses a significant challenge with respect to strain coverage. There are more than seventy-seven known capsule serotypes and eight O-antigen serotypes, and it is not clear which are the most prevalent or associated with pathogenesis. In addition, the limited number of monoclonal antibodies targeting conserved epitopes within LPS have no reported protective effect (Brade et al. 2001, J Endotoxin Res, 7(2):119-24).

Thus, there is a great need to identify and develop antibodies that have protective effect against Klebsiella, (e.g., K. pneumoniae), especially antibiotic resistant Klebsiella, infections.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides K. pneumoniae O2 binding proteins, e.g., antibodies or antigen binding fragments thereof, and methods of treating Klebsiella infections using K. pneumoniae O2 binding proteins.

In one instance provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen, wherein the antigen binding protein induces opsonophagocytic killing (OPK) of Klebsiella. In one instance, the antigen binding protein induces OPK of O1 serotype Klebsiella and O2 serotype Klebsiella. In one instance, the antigen binding protein induces OPK of O2 serotype Klebsiella, but does not induce OPK of O1 serotype Klebsiella.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen protects mice from a lethal Klebsiella challenge.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen neutralizes lipopolysaccharide (LPS). In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen inhibits, reduces, or prevents NF-kB activation induced by LPS. In one instance, the antigen binding protein inhibits, reduces, or prevents NF-kB activation induced by both Klebsiella pneumoniae O1 LPS and Klebsiella pneumoniae O2 LPS. In one instance, the antigen binding protein inhibits, reduces, or prevents NF-kB activation induced by Klebsiella pneumoniae O2 LPS, but does not inhibit, reduce, or prevent NF-kB activation induced by Klebsiella pneumoniae O1 LPS.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen: (i) neutralizes O1 and/or O2 LPS and induces OPK of O2 serotype Klebsiella but does not induce OPK of O1 serotype Klebsiella; (ii) neutralizes O1 and/or O2 LPS and induces OPK of O1 serotype Klebsiella and O2 serotype Klebsiella; or (iii) does not neutralize O1 LPS and induces OPK of O2 serotype Klebsiella but does not induce OPK of O1 serotype Klebsiella.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen induces OPK of K. pneumoniae, K. oxytoca, K. granulomatis, K. ozaenae, K. rhinosclermoatis and/or K. planticola. In one instance, the antigen binding protein induces OPK of K. pneumoniae.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen also binds to Klebsiella pneumoniae O1 antigen.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen a) induces OPK in a multi-drug resistant Klebsiella, b) protects mice from a lethal multi-drug resistant Klebsiella challenge, or c) induces OPK of a multi-drug resistant Klebsiella and protects mice from a lethal multi-drug resistant Klebsiella challenge. In one instance, the multi-drug resistant Klebsiella is strain Kp961842 or Kp977778 (both of which are ST258 strains). In one instance, the multi-drug resistant Klebsiella is a strain listed in one of rows 1-226 of Table 8.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen renders a multi-drug resistant K. pneumoniae strain sensitive to at least one antibiotic.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen a) induces OPK in a Klebsiella that is susceptible to antibiotics, b) protects mice from a lethal Klebsiella challenge, wherein the Klebsiella is susceptible to antibiotics, or c) induces OPK in a Klebsiella that is susceptible to antibiotics and protects mice from a lethal Klebsiella challenge, wherein the Klebsiella is susceptible to antibiotics. In one instance, the Klebsiella is a strain listed in one of rows 227-254 of Table 8.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen binds to gml− Klebsiella. In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen binds to gml+ Klebsiella.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen binds to the D-Galactan I domain of K. pneumoniae O2 antigen.

In one instance, provided herein is an isolated antigen binding protein that specifically binds Klebsiella pneumoniae O2 antigen, wherein the antigen binding protein comprises a heavy chain variable region (VH) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:8 and/or a light chain variable region (VL) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:9. In one instance, the antigen binding protein thereof comprises a VH comprising SEQ ID NO:8 and a VL comprising SEQ ID NO:9.

In one instance, provided herein is an isolated antigen binding protein that specifically binds Klebsiella pneumoniae O2 antigen, wherein the antigen binding protein comprises a heavy chain variable region (VH) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:202 and/or a light chain variable region (VL) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:203. In one instance, the antigen binding protein thereof comprises a VH comprising SEQ ID NO:202 and a VL comprising SEQ ID NO:203.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VH comprising SEQ ID NO:8.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VH comprising SEQ ID NO:202.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VL comprising SEQ ID NO:9.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VL comprising SEQ ID NO:203.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a set of Complementarity-Determining Regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprise the amino acid sequences of: SEQ. ID. NOs: 10-13, 14 or 15, and 16, respectively; SEQ. ID. NOs: 19-22, 23 or 24, and 25, respectively; SEQ. ID. NOs: 28-31, 32 or 33, and 34, respectively; SEQ. ID. NOs: 37-40, 41 or 42, and 43, respectively; SEQ. ID. NOs: 46-49, 50 or 51, and 52, respectively; SEQ. ID. NOs: 166-168, 175, 176 or 177, and 178, respectively; SEQ. ID. NOs: 169-171, 179, 180 or 181, and 182, respectively; SEQ. ID. NOs: 55-58, 59 or 60, and 61, respectively; SEQ. ID. NOs: 64-67, 68 or 69, and 70, respectively; SEQ. ID. NOs: 73-78, respectively; SEQ. ID. NOs: 82-85, 86 or 87, and 88, respectively; SEQ. ID. NOs: 91-94, 95 or 96, and 97, respectively; SEQ. ID. NOs: 100-103, 104 or 105, and 106, respectively; SEQ. ID. NOs: 109-112, 113 or 114, and 115, respectively; SEQ. ID. NOs: 118-121, 122 or 123, and 124, respectively; SEQ. ID. NOs: 127-130, 131 or 132, and 133, respectively; SEQ. ID. NOs: 172-174, 183, 184 or 185, and 186, respectively; or SEQ ID NOs: 109-111, 199, 112-115, 200 and 201.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen, wherein the antigen binding protein comprises a VH and VL at least 95%, 96%, 97%, 98%, or 99% identical to: SEQ. ID. NO: 17 and SEQ ID NO:18, respectively; SEQ. ID. NO: 26 and SEQ ID NO:27, respectively; SEQ. ID. NO: 35 and SEQ ID NO:36, respectively; SEQ. ID. NO: 44 and SEQ ID NO:45, respectively; SEQ. ID. NO: 53 and SEQ ID NO:54, respectively; SEQ. ID. NO: 187 and SEQ ID NO:190, respectively; SEQ. ID. NO: 188 and SEQ ID NO:191, respectively; SEQ. ID. NO: 62 and SEQ ID NO:63, respectively; SEQ. ID. NO: 71 and SEQ ID NO:72, respectively; SEQ. ID. NO: 80 and SEQ ID NO:81, respectively; SEQ. ID. NO: 89 and SEQ ID NO:90, respectively; SEQ. ID. NO: 98 and SEQ ID NO:99, respectively; SEQ. ID. NO: 107 and SEQ ID NO:108, respectively; SEQ. ID. NO: 116 and SEQ ID NO:117, respectively; SEQ. ID. NO: 125 and SEQ ID NO:126, respectively; SEQ. ID. NO: 134 and SEQ ID NO:135, respectively; SEQ. ID. NO:189 and SEQ ID NO:192, respectively; or SEQ ID NOs 116 and 202-205.

In one instance, the antigen binding protein comprises a VH and a VL comprising: SEQ. ID. NO: 17 and SEQ ID NO:18, respectively; SEQ. ID. NO: 26 and SEQ ID NO:27, respectively; SEQ. ID. NO: 35 and SEQ ID NO:36, respectively; SEQ. ID. NO: 44 and SEQ ID NO:45, respectively; SEQ. ID. NO: 53 and SEQ ID NO:54, respectively; SEQ. ID. NO: 187 and SEQ ID NO:190, respectively; SEQ. ID. NO: 188 and SEQ ID NO:191, respectively; SEQ. ID. NO: 62 and SEQ ID NO:63, respectively; SEQ. ID. NO: 71 and SEQ ID NO:72, respectively; SEQ. ID. NO: 80 and SEQ ID NO:81, respectively; SEQ. ID. NO: 89 and SEQ ID NO:90, respectively; SEQ. ID. NO: 98 and SEQ ID NO:99, respectively; SEQ. ID. NO: 107 and SEQ ID NO:108, respectively; SEQ. ID. NO: 116 and SEQ ID NO:117, respectively; SEQ. ID. NO: 125 and SEQ ID NO:126, respectively; SEQ. ID. NO: 134 and SEQ ID NO:135, respectively; SEQ. ID. NO: 189 and SEQ ID NO:192, respectively; SEQ ID NOs 116 and 202-205; SEQ ID NO:273 and SEQ ID NO:247, respectively; SEQ ID NO:273 and SEQ ID NO:257, respectively; SEQ ID NO:273 and SEQ ID NO:217, respectively; SEQ ID NO:273 and SEQ ID NO:227, respectively; SEQ ID NO:274 and SEQ ID NO:247, respectively; SEQ ID NO:274 and SEQ ID NO:257, respectively; SEQ ID NO:274 and SEQ ID NO:217, respectively; and SEQ ID NO:274 and SEQ ID NO:227, respectively.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VH comprising SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO: 35, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98, SEQ ID NO:107, SEQ ID NO:116, SEQ ID NO:125, SEQ ID NO:134, SEQ ID NO:189; or SEQ ID NO: 116; SEQ ID NO:202; SEQ ID NO:213; SEQ ID NO:223; SEQ ID NO:233; SEQ ID NO:243; SEQ ID NO:253; SEQ ID NO:263; SEQ ID NO:273; or SEQ ID NO:274.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprising a VL comprising SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO: 36, SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:99, SEQ ID NO:108, SEQ ID NO:117, SEQ ID NO:126, SEQ ID NO:135, SEQ ID NO:192; SEQ ID NO: 203; SEQ ID NO:204; SEQ ID NO:205; SEQ ID NO:217; SEQ ID NO:227; SEQ ID NO:237; SEQ ID NO:247; SEQ ID NO:257; or SEQ ID NO:267.

In one instance, provided herein is an isolated antigen binding protein that specifically binds to the same epitope in the Klebsiella pneumoniae O2 antigen as an antibody comprising a VH and a VL comprising: SEQ. ID. NO: 17 and SEQ ID NO:18, respectively; SEQ. ID. NO: 26 and SEQ ID NO:27, respectively; SEQ. ID. NO: 35 and SEQ ID NO:36, respectively; SEQ. ID. NO: 44 and SEQ ID NO:45, respectively; SEQ. ID. NO: 53 and SEQ ID NO:54, respectively; SEQ. ID. NO: 187 and SEQ ID NO:190, respectively; SEQ. ID. NO: 188 and SEQ ID NO:191, respectively; SEQ. ID. NO: 62 and SEQ ID NO:63, respectively; SEQ. ID. NO: 71 and SEQ ID NO:72, respectively; SEQ. ID. NO: 80 and SEQ ID NO:81, respectively; SEQ. ID. NO: 89 and SEQ ID NO:90, respectively; SEQ. ID. NO: 98 and SEQ ID NO:99, respectively; SEQ. ID. NO: 107 and SEQ ID NO:108, respectively; SEQ. ID. NO: 116 and SEQ ID NO:117, respectively; SEQ. ID. NO: 125 and SEQ ID NO:126, respectively; SEQ. ID. NO: 134 and SEQ ID NO:135, respectively; SEQ. ID. NO: 189 and SEQ ID NO:192; or SEQ ID NOs 116 and 202-205.

In one instance, provided herein is an isolated antigen binding protein that competitively inhibits the binding to Klebsiella pneumoniae O2 antigen of an antibody comprising a VH and a VL comprising: SEQ. ID. NO: 17 and SEQ ID NO:18, respectively; SEQ. ID. NO: 26 and SEQ ID NO:27, respectively; SEQ. ID. NO: 35 and SEQ ID NO:36, respectively; SEQ. ID. NO: 44 and SEQ ID NO:45, respectively; SEQ. ID. NO: 53 and SEQ ID NO:54, respectively; SEQ. ID. NO: 187 and SEQ ID NO:190, respectively; SEQ. ID. NO: 188 and SEQ ID NO:191, respectively; SEQ. ID. NO: 62 and SEQ ID NO:63, respectively; SEQ. ID. NO: 71 and SEQ ID NO:72, respectively; SEQ. ID. NO: 80 and SEQ ID NO:81, respectively; SEQ. ID. NO: 89 and SEQ ID NO:90, respectively; SEQ. ID. NO: 98 and SEQ ID NO:99, respectively; SEQ. ID. NO: 107 and SEQ ID NO:108, respectively; SEQ. ID. NO: 116 and SEQ ID NO:117, respectively; SEQ. ID. NO: 125 and SEQ ID NO:126, respectively; SEQ. ID. NO: 134 and SEQ ID NO:135, respectively; SEQ. ID. NO: 189 and SEQ ID NO:192; or SEQ ID NOs 116 and 202-205.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is murine, non-human, humanized, chimeric, resurfaced, or human.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is an antibody. In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is an antigen binding fragment of an antibody. In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, or an antigen binding fragment thereof. In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen comprises a Fab, Fab′, F(ab′)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGACH2, minibody, F(ab′)3, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) binds to Klebsiella O2 antigen with an affinity constant of about 4.5E-09 or about 7.8E-09M. In one instance, the binding affinity is measured by octet binding, flow cytometry, Biacore, KinExa, or radioimmunoassay. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) induces OPK of Klebsiella. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) induces OPK of O1 serotype Klebsiella and O2 serotype Klebsiella. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) induces OPK of O2 serotype Klebsiella, but does not induce OPK of O1 serotype Klebsiella. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) protects mice from a lethal Klebsiella challenge.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) neutralizes LPS. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof) inhibits, reduces, or prevents NF-kB activation induced by LPS. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) inhibits, reduces, or prevents NF-kB activation induced by both Klebsiella pneumoniae O1 LPS and Klebsiella pneumoniae O2 LPS. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) inhibits, reduces, or prevents NF-kB activation induced by Klebsiella pneumoniae O2 LPS, but does not inhibit, reduce, or prevent NF-kB activation induced by Klebsiella pneumoniae O1 LPS. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) does not neutralize O1 LPS and induces OPK of O2 serotype Klebsiella but does not induce OPK of O1 serotype Klebsiella.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen): (i) neutralizes O1 and/or O2 LPS and induces OPK of O2 serotype Klebsiella but does not induce OPK of O1 serotype Klebsiella; (ii) neutralizes O1 and/or O2 LPS and induces OPK of O1 serotype Klebsiella and O2 serotype Klebsiella; or (iii) does not neutralize O1 LPS and induces OPK of O2 serotype Klebsiella but does not induce OPK of O1 serotype Klebsiella.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) induces OPK of K. pneumoniae, K oxytoca, K. granulomatis, K. ozaenae, K. rhinosclermoatis and/or K. planticola. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) induces OPK of K. pneumoniae.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) binds to Klebsiella pneumoniae O1 antigen. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen): a) induces OPK in a multi-drug resistant Klebsiella, b) protects mice from a lethal multi-drug resistant Klebsiella challenge, or c) induces OPK of a multi-drug resistant Klebsiella and protects mice from a lethal multi-drug resistant Klebsiella challenge. In one instance, the multi-drug resistant Klebsiella is strain Kp961842 or Kp977778. In one instance, the multi-drug resistant Klebsiella is a strain listed in one of rows 1-226 of Table 8. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) renders a multi-drug resistant K. pneumoniae strain sensitive to at least one antibiotic.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen): a) induces OPK in a Klebsiella that is susceptible to antibiotics, b) protects mice from a lethal Klebsiella challenge, wherein the Klebsiella is susceptible to antibiotics, or c) induces OPK in a Klebsiella that is susceptible to antibiotics and protects mice from a lethal Klebsiella challenge, wherein the Klebsiella is susceptible to antibiotics. In one instance, the Klebsiella is a strain listed in one of rows 227-254 of Table 8.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) binds to gml− Klebsiella. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) binds to gml+ Klebsiella. In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) binds to the D-Galactan I domain of K. pneumoniae O2 antigen.

In one instance, the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) comprises a heavy chain immunoglobulin constant domain selected from the group consisting of: (a) an IgA constant domain; (b) an IgD constant domain; (c) an IgE constant domain; (d) an IgG1 constant domain; (e) an IgG2 constant domain; (f) an IgG3 constant domain; (g) an IgG4 constant domain; and (h) an IgM constant domain.

In one instance, provided herein is an isolated nucleic acid molecule encoding an antigen binding protein provided herein, including e.g., an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen. In one instance, the nucleic acid molecule is operably linked to a control sequence.

In one instance, provided herein is a vector comprising a nucleic acid molecule provided herein.

In one instance, provided herein is a host cell transformed with a nucleic acid molecule provided herein or a vector provided herein. In one instance, the host cell is a mammalian host cell. In one instance, the host cell is a HEK293 cell, CHO cell, COS-7 cell, a HeLa cell, a NS0 murine myeloma cell, or a PER.C6® human cell.

In one instance, provided herein is a hybridoma producing an antigen binding protein provided herein, including e.g., an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen. In one instance provided herein is an isolated host cell producing an antigen binding protein provided herein, including e.g., an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen. In one instance, provided herein is a method of making an antigen binding protein provided herein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) comprising (a) culturing a host cell expressing the antigen binding protein or culturing a host cell provided herein or a hybridoma provided herein; and (b) isolating the antigen binding protein thereof from the cultured host cell or hybridoma. In one instance, provided herein is an antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) produced using a method provided herein.

In one instance, provided herein is a pharmaceutical composition comprising an antigen binding provided herein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) and a pharmaceutically acceptable excipient. In one instance, the pharmaceutically acceptable excipient is a preservative, stabilizer, or antioxidant. In one instance, the pharmaceutical composition is for use as a medicament.

In one instance, the antigen binding protein provided herein or the pharmaceutical composition provided herein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) further comprises a labeling group or an effector group. In one instance, the labeling group is selected from the group consisting of: isotopic labels, magnetic labels, redox active moieties, optical dyes, biotinylated groups, fluorescent moieties such as biotin signaling peptides, Green Fluorescent Proteins (GFPs), blue fluorescent proteins (BFPs), cyan fluorescent proteins (CFPs), yellow fluorescent proteins (YFPs), polypeptide epitopes recognized by a secondary reporter such as histidine peptide (his), hemagglutinin (HA), gold binding peptide, and Flag. In one instance, the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic and a chemotherapeutic agent.

In one instance, provided herein is the use of an antigen binding protein or pharmaceutical composition provided herein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) for treating a condition associated with a Klebsiella infection.

In one instance, provided herein is a method for treating, preventing, or ameliorating a condition associated with a Klebsiella infection in a subject in need thereof comprising administering to the subject an effective amount of an antigen binding protein provided herein or a pharmaceutical composition provided herein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen).

In one instance, provided herein is a method for inhibiting the growth of Klebsiella, or reducing the number of Klebsiella in a subject infected with Klebsiella comprising administering to a subject in need thereof an antigen binding protein provided herein or a pharmaceutical composition provided herein.

In one instance, provided herein is a method for treating, preventing, or ameliorating a condition associated with a Klebsiella infection in a subject in need thereof comprising administering to the subject an effective amount of antigen binding protein that that specifically binds to Klebsiella pneumoniae O2 antigen.

In one instance, provided herein is a method for inhibiting the growth of Klebsiella, or reducing the number of Klebsiella in a subject infected with Klebsiella comprising administering to a subject an effective amount of an antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen.

In one instance, the Klebsiella is antibiotic-resistant. In one instance, the Klebsiella is resistant to cephalosporin, quinolone, carbapnem, meroprem, fluoroquinolone, tetracycline, chloramphenicol, trimethoprim, sulfonamide, and/or colistin.

In one instance, provided herein is a method for sensitizing an antibiotic-resistant Klebsiella strain to antibiotics comprising contacting the antibody-resistant Klebsiella strain with an antigen binding protein that that specifically binds to Klebsiella pneumoniae O2 antigen.

In one instance, the method further comprises administering an antibiotic. In one instance, the antigen binding protein and the antibiotic provide a synergistic therapeutic effect.

In one instance, provided herein is a method of preventing or treating a condition associated with a Klebsiella infection in a subject infected with an antibiotic-resistant Klebsiella strain, comprising co-administering to a subject an antibiotic and an antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen, wherein the co-administration provides a therapeutic effect greater than the sum of the individual effects of administration of equal molar quantities of the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof) or the antibiotic. In one instance, the therapeutic effect results in greater percent survival than the additive percent survival of subjects to which only one of the antigen binding protein (e.g. an antibody or antigen-binding fragment thereof that specifically binds to Klebsiella pneumoniae O2 antigen) or the antibiotic was administered. In one instance, the antibiotic is meropenem, carbapenems, fluoroquinolone, tetracycline, chloramphenicol, trimethoprim, sulfonamide, and/or colistin. In one instance, the antigen binding protein also specifically binds Klebsiella pneumoniae O1 antigen.

In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is an antibody or antigen binding fragment thereof. In one instance, the antigen binding protein that specifically binds to Klebsiella pneumoniae O2 antigen is an antigen binding protein provided herein or a pharmaceutical composition provided herein.

In one instance, the Klebsiella is K. pneumoniae, K. oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis and/or K. granulomatis. In one instance, the Klebsiella is K. pneumoniae.

In one instance, the condition is selected from the group consisting of pneumonia, urinary tract infection, septicemia/sepsis, neonatal septicemia/sepsis, diarrhea, soft tissue infection, infection following an organ transplant, surgery infection, wound infection, lung infection, pyogenic liver abscesses (PLA), endophthalmitis, meningitis, necrotizing meningitis, ankylosing spondylitis, and spondyloarthropathies. In one instance, the condition is a nosocomial infection.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIGS. 1A-B show the expansion of the O2 LPS serotype in multi-drug resistant (MDR) strains of Klebsiella pneumoniae. FIG. 1A shows 0 serotype determining antigens for Klebsiella pneumoniae O1/O2 LPS and a western blot analysis of seven purified Klebsiella LPS serotypes probed with an isolated anti-O2 monoclonal antibody (mAb). FIG. 1B shows the prevalence of the O1 and O2 serotypes in recent Klebsiella pneumoniae clinical isolates.

FIGS. 2A-C show the characterization of anti-O2 LPS monoclonal antibodies (mAbs) by enzyme-linked immunosorbent assay (ELISA). FIG. 2A shows the binding of representative mAbs from each antibody class to O1 LPS. FIG. 2B shows the binding of representative mAbs from each class to O2 LPS. FIG. 2C shows the half maximal effective concentration (EC₅₀) of each mAb binding to LPS-O1 or LPS-O2.

FIGS. 3A-3O show the results of an octet binding assay with anti-O2 monoclonal antibodies (mAbs). FIGS. 3A-3F show sensorgram traces of anti-O2 mAbs interacting with O1 LPS. FIGS. 3G-3L show sensorgram traces of anti-O2 mAbs interacting with O2 LPS. FIGS. 3M-3O show affinity measurements of the Class III mAbs KPN42 and KPN179.

FIGS. 4A-4F show LPS neutralization assays performed with selected monoclonal antibodies (mAbs). The % neutralization of the mAbs against LPS-O1 is shown in FIGS. 4A-4C, and the % neutralization of the mAbs against LPS-O2 is shown in FIGS. 4D-4F.

FIGS. 5A-5B show the opsonophagocytic killing (OPK) activity of selected anti-O2 monoclonal antibodies (mAbs). The OPK of these mAbs against an 01 strain of K. pneumoniae is shown in FIG. 5A, and the OPK of these mAbs against an O2 strain of K. pneumoniae is shown in FIG. 5B.

FIGS. 6A-6B show the protection conferred by Class III monoclonal antibodies (mAbs) in lethal pneumonia models. The % survival of mice challenged with K. pneumoniae Carbanpenemase (KPC) strain Kp961842_O2 is shown in FIG. 6A, and the % survival of mice challenged with KPC strain Kp977778_O2 is shown in FIG. 6B.

FIGS. 7A-7B show shows that the anti-O2 LPS monoclonal antibodies (mAbs) KPN42 and KPN179 have strong synergy with meropenem in a lethal pneumonia model, as measured by the % survival of mice at various time points post challenge.

FIGS. 8A-8B show that select anti-O2 monoclonal antibodies (mAbs) protect mice for up to 6 hours post infection in conjunction with Meropenem. For mice treated with KPN42 in conjunction with meropenem, the % survival at various time points post challenge is shown in FIG. 8A. For mice treated with KPN179 in conjunction with meropenem, the % survival at various time points post challenge is shown in FIG. 8B.

FIGS. 9A-9C show the sequence optimization of KPN42 and KPN179. The binding of optimized versions of KPN42 and KPN179 to O2 LPS is shown in the graph in FIG. 9C.

FIGS. 10A-C shows the protection conferred by Class III monoclonal antibodies KPS44 and KPS44v2017 in an opsonophagocytic killing (OPK) assay against an O2 strain of K. pneumoniae (FIG. 10B) and lethal pneumonia models against the KPS ST258 O2 strain 961842 (FIGS. 10A and 10C).

FIG. 11A shows the binding affinities from an octet binding assay with anti-O2 monoclonal antibodies (mAbs). FIG. 11B shows the protection conferred by Class III monoclonal antibodies KPS44, KPS44v2017, KPS44-G2, and KPS44-G3 in an opsonophagocytic killing (OPK) assay against an O2 strain of K. pneumoniae

DETAILED DESCRIPTION OF THE INVENTION

The data provided herein shows that a high prevalence of multidrug resistant Klebsiella pneumoniae strains are of the O2 serotype (see Example 1). Thus, there is a particularly great need to identify and develop antibodies that have protective effect against Klebsiella of the O2 serotype. Accordingly, the present disclosure provides isolated binding proteins, including antibodies or antigen binding fragments thereof, that bind to Klebsiella pneumoniae O2 antigen. Related polynucleotides, vectors, host cells, and pharmaceutical compositions comprising the Klebsiella pneumoniae O2 binding proteins, including antibodies or antigen binding fragments thereof, are also provided. Also provided are methods of making and using the O2 binding proteins, including antibodies or antigen binding fragments, disclosed herein. The present disclosure also provides methods of preventing and/or treating a condition associated with a Klebsiella infection (e.g., K. pneumoniae such as O2 serotype K. pneumoniae) by administering the O2 binding proteins, including antibodies or antigen binding fragments, disclosed herein.

In order that the present disclosure can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

I. Definitions

The terms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “an antigen binding protein” is understood to represent one or more antigen binding proteins. The terms “a” (or “an”), as well as the terms “one or more,” and “at least one” can be used interchangeably herein. Furthermore, “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of,” and/or “consisting essentially of” are also provided.

The term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is ±10%.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

The term “antigen binding protein” refers to a molecule comprised of one or more polypeptides that recognizes and specifically binds to a target, e.g., K. pneumoniae O2 antigen, such as an anti-O2 antibody or antigen-binding fragment thereof.

The term “antibody” means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term “antibody” encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antibody, and any other modified immunoglobulin molecule so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” or “antibody fragment thereof” refers to a portion of an intact antibody. An “antigen-binding fragment” or “antigen-binding fragment thereof” refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment can contain the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, scFvs, and single chain antibodies.

It is possible to take monoclonal and other antibodies or fragments thereof and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules or fragments thereof that retain the specificity of the original antibody or fragment. Such techniques can involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A, or EP-A-239400, and a large body of subsequent literature. A hybridoma or other cell producing an antibody can be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies or fragments thereof produced.

Further techniques available in the art of antibody engineering have made it possible to isolate human and humanized antibodies or fragments thereof. For example, human hybridomas can be made as described by Kontermann and Sefan. Antibody Engineering, Springer Laboratory Manuals (2001). Phage display, another established technique for generating antigen binding proteins has been described in detail in many publications such as Kontermann and Sefan. Antibody Engineering, Springer Laboratory Manuals (2001) and WO92/01047. Transgenic mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies to human antigens.

Synthetic antibodies or fragments thereof can be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et al. J. Mol. Biol. (2000) 296, 57-86 or Krebs et al. Journal of Immunological Methods 254 2001 67-84.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL, and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989), McCafferty et al (1990) Nature, 348, 552-554) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fab fragments; and (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); Fv or scFv molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domain may also be made (S. Hu et al, Cancer Res., 56, 3055-3061, 1996).

The phrase “effector function” refers to the activities of antibodies that result from the interactions of their Fc components with Fc receptors or components of complement. These activities include, for example, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell phagocytosis (ADCP). Thus an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) with altered effector function refers to an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) that contains an alteration in an Fc region (e.g., amino acid substitution, deletion, or addition or change in oligosaccharide) that changes the activity of at least one effector function (e.g., ADCC, CDC, and/or ADCP). An antigen binding protein (e.g., an antibody or antigen binding fragment thereof) with improved effector function refers to an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) that contains an alteration in an Fc region (e.g., amino acid substitution, deletion, or addition or change in oligosaccharide) that increases the activity of at least one effector function (e.g., ADCC, CDC, and/or ADCP).

The term “specific” can be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the antigen binding protein carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

By “specifically binds” it is generally meant that an antigen binding protein including an antibody or antigen binding fragment thereof binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope via its antigen binding domain more readily than it would bind to a random, unrelated epitope. As used herein, an antigen binding protein that “specifically binds” to Klebsiella pneumoniae O2 antigen may or may not also bind to other Klebsiella pneumoniae 0 antigens, including, for example, Klebsiella pneumoniae O1 antigen. In some embodiments, the antigen binding proteins disclosed herein specifically bind both Klebsiella pneumoniae O2 antigen and Klebsiella pneumoniae O1 antigen, while in other embodiments the antigen binding proteins disclosed herein specifically bind Klebsiella pneumoniae O2 antigen but do not specifically bind Klebsiella pneumoniae O1 antigen.

“Affinity” is a measure of the intrinsic binding strength of a ligand binding reaction. For example, a measure of the strength of the antibody (Ab)-antigen (Ag) interaction is measured through the binding affinity, which may be quantified by the dissociation constant, k_d. The dissociation constant is the binding affinity constant and is given by:

$K_{d} = \begin{matrix} [Ab] [Ag] \\ [AbAg complex] \end{matrix}$

Affinity may, for example, be measured using a BIAcore®, a KinExA affinity assay, flow cytometry, and/or radioimmunoassay.

“Potency” is a measure of pharmacological activity of a compound expressed in terms of the amount of the compound required to produce an effect of given intensity. It refers to the amount of the compound required to achieve a defined biological effect; the smaller the dose required, the more potent the drug. Potency of an antigen binding protein that binds O2 can, for example, be determined using an OPK assay, as described herein.

“Opsonophagocytic killing” or “OPK” refers to the death of a cell, e.g., a Klebsiella, that occurs as a result of phagocytosis by an immune cell. OPK activity is measured according to the bioluminescent assay used in Example 8. An antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) can induce OPK where the percentage of killing is 40% or greater. An antigen binding protein (e.g., an antibody or antigen-binding fragment thereof) can strongly induce OPK where the percentage of killing is 80% or greater.

An antigen binding protein including an antibody or antigen binding fragment thereof is said to competitively inhibit binding of a reference antibody or antigen binding fragment thereof to a given epitope or “compete” with a reference antibody or antigen binding fragment if it blocks, to some degree, binding of the reference antibody or antigen binding fragment to the epitope. Competitive inhibition can be determined by any method known in the art, for example, competition ELISA assays. A binding molecule can be said to competitively inhibit binding of the reference antibody or antigen binding fragment to a given epitope or compete with a reference antibody or antigen binding fragment thereof by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

The term “compete” when used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) means competition between antigen binding proteins as determined by an assay in which the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) under test prevents or inhibits specific binding of a reference antigen binding protein (e.g., a ligand, or a reference antibody) to a common antigen (e.g., an O2 polysaccharide or a fragment thereof). Numerous types of competitive binding assays can be used, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 92:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase direct labeled assay, solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antigen binding protein.

Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test antigen binding protein. Usually the test antigen binding protein is present in excess. Antigen binding proteins identified by competition assay (competing antigen binding proteins) include antigen binding proteins binding to the same epitope as the reference antigen binding proteins and antigen binding proteins binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antigen binding protein for steric hindrance to occur. Usually, when a competing antigen binding protein is present in excess, it will inhibit specific binding of a reference antigen binding protein to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

Antigen binding proteins, antibodies or antigen binding fragments thereof disclosed herein can be described or specified in terms of the epitope(s) or portion(s) of an antigen, e.g., a target polypeptide that they recognize or specifically bind. For example, the portion of O2 that specifically interacts with the antigen binding domain of the antigen binding polypeptide or fragment thereof disclosed herein is an “epitope”. Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. A conformational epitope can be composed of discontinuous sections of the antigen's amino acid sequence. A linear epitope is formed by a continuous sequence of amino acids from the antigen. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and can have specific three dimensional structural characteristics, and/or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35 amino acids in a unique spatial conformation. Epitopes can be determined using methods known in the art.

Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

As used herein, the term “polypeptide” refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. As used herein the term “protein” is intended to encompass a molecule comprised of one or more polypeptides, which can in some instances be associated by bonds other than amide bonds. On the other hand, a protein can also be a single polypeptide chain. In this latter instance the single polypeptide chain can in some instances comprise two or more polypeptide subunits fused together to form a protein. The terms “polypeptide” and “protein” also refer to the products of post-expression modifications, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide or protein can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

The term “isolated” refers to the state in which antigen binding proteins of the disclosure, or nucleic acid encoding such binding proteins, will generally be in accordance with the present disclosure. Isolated proteins and isolated nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in vivo. Proteins and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated—for example the proteins will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Antigen binding proteins may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NS0 (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

A polypeptide, antigen binding protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, antigen binding protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antigen binding proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antigen binding protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

A “recombinant” polypeptide, protein or antibody refers to a polypeptide or protein or antibody produced via recombinant DNA technology. Recombinant polypeptides, proteins and antibodies expressed in host cells are considered isolated for the purpose of the present disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

Also included in the present disclosure are fragments, variants, or derivatives of polypeptides, and any combination thereof. The term “fragment” when referring to polypeptides and proteins of the present disclosure include any polypeptides or proteins which retain at least some of the properties of the reference polypeptide or protein. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments.

The term “variant” as used herein refers to an antibody or polypeptide sequence that differs from that of a parent antibody or polypeptide sequence by virtue of at least one amino acid modification. Variants of antibodies or polypeptides of the present disclosure include fragments, and also antibodies or polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

The term “derivatives” as applied to antibodies or polypeptides refers to antibodies or polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide or protein. An example of a “derivative” antibody is a fusion or a conjugate with a second polypeptide or another molecule (e.g., a polymer such as PEG, a chromophore, or a fluorophore) or atom (e.g., a radioisotope).

The terms “polynucleotide” or “nucleotide” as used herein are intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), complementary DNA (cDNA), or plasmid DNA (pDNA). In certain aspects, a polynucleotide comprises a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)).

The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA, cDNA, or RNA fragments, present in a polynucleotide. When applied to a nucleic acid or polynucleotide, the term “isolated” refers to a nucleic acid molecule, DNA or RNA, which has been removed from its native environment, for example, a recombinant polynucleotide encoding an antigen binding protein contained in a vector is considered isolated for the purposes of the present disclosure. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

As used herein, the term “host cell” refers to a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid. Host cells can be a prokaryotic cells (e.g., E. coli), or alternatively, the host cells can be eukaryotic, for example, fungal cells (e.g., yeast cells such as Saccharomyces cerivisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g., Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS-7, NIH-3T3, a NS0 murine myeloma cell, a PER.C6® human cell, a Chinese hamster ovary (CHO) cell or a hybridoma).

The term “amino acid substitution” refers to replacing an amino acid residue present in a parent sequence with another amino acid residue. An amino acid can be substituted in a parent sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a “substitution at position X” or “substitution at position X” refer to the substitution of an amino acid present at position X with an alternative amino acid residue. In some embodiments, substitution patterns can described according to the schema AXY, wherein A is the single letter code corresponding to the amino acid naturally present at position X, and Y is the substituting amino acid residue. In other aspects, substitution patterns can described according to the schema XY, wherein Y is the single letter code corresponding to the amino acid residue substituting the amino acid naturally present at position X.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

Non-conservative substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

Other substitutions can be readily identified by workers of ordinary skill. For example, for the amino acid alanine, a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, omithine, or D-ornithine. Generally, substitutions in functionally important regions that can be expected to induce changes in the properties of isolated polypeptides are those in which (i) a polar residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine. The likelihood that one of the foregoing non-conservative substitutions can alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions can accordingly have little or no effect on biological properties.

The term “amino acid insertion” refers to introducing a new amino acid residue between two amino acid residues present in the parent sequence. An amino acid can be inserted in a parent sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly as used herein, the phrases “insertion between positions X and Y,” “insertion between IMGT positions X and Y,” or “insertion between Kabat positions X and Y,” wherein X and Y correspond to amino acid positions (e.g., a cysteine amino acid insertion between positions 239 and 240), refers to the insertion of an amino acid between the X and Y positions, and also to the insertion in a nucleic acid sequence of a codon encoding an amino acid between the codons encoding the amino acids at positions X and Y. Insertion patterns can be described according to the schema AXins, wherein A is the single letter code corresponding to the amino acid being inserted, and X is the position preceding the insertion.

The term “percent sequence identity” or “percent identity” between two polynucleotide or polypeptide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence. The percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences can be accomplished using readily available software programs. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

“Specific binding member” describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present disclosure is concerned with antigen-antibody type reactions.

The term “IgG” as used herein refers to a polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans this class comprises IgG1, IgG2, IgG3, and IgG4. In mice this class comprises IgG1, IgG2a, IgG2b, and IgG3.

The term “antigen binding domain” describes the part of an antibody that comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains (e.g. a so-called Fd antibody fragment consisting of a VH domain). An antigen binding domain may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

The term “antigen binding protein fragment” or “antibody fragment” refers to a portion of an intact antigen binding protein or antibody and refers to the antigenic determining variable regions of an intact antigen binding protein or antibody. It is known in the art that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab′, F(ab′)₂, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, “monoclonal antibody” refers to such antibodies made in any number of ways including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

The term “human antibody” refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides. The term “humanized antibody” refers to an antibody derived from a non-human (e.g., murine) immunoglobulin, which has been engineered to contain minimal non-human (e.g., murine) sequences.

The term “chimeric antibody” refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

The term “antibody binding site” refers to a region in the antigen (e.g., O2) comprising a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds. Thus, the antibody binding site can contain additional areas in the antigen which are beyond the epitope and which can determine properties such as binding affinity and/or stability, or affect properties such as antigen enzymatic activity or dimerization. Accordingly, even if two antibodies bind to the same epitope within an antigen, if the antibodies establish distinct intermolecular contacts with amino acids outside of the epitope, such antibodies are considered to bind to distinct antibody binding sites.

The IMGT numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g. Lefranc, M.-P. et al. Dev. Comp. Immunol. 27: 55-77 (2003)).

The phrases “amino acid position numbering as in Kabat,” “Kabat position,” and grammatical variants thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FW or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FW residue 82.

The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The IMGT (Lefranc, M.-P. et al. Dev. Comp. Immunol. 27: 55-77 (2003)) classification of CDRs can also be used.

The term “EU index as in Kabat” refers to the numbering system of the human IgG1 EU antibody described in Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). All amino acid positions referenced in the present application refer to IMGT unique numbering unless indicated otherwise. For example, C105 is defined according to IMGT unique numbering. For example, KPN42-C105 and KPN179-C105 correspond to Kabat position 89.

The terms “Fc domain,” “Fc Region,” and “IgG Fc domain” as used herein refer to the portion of an immunoglobulin, e.g., an IgG molecule, that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and binding sites for complement and Fc receptors, including the FcRn receptor. For example, an Fc domain contains the entire second constant domain CH2 (residues at EU positions 231-340 of human IgG1) and the third constant domain CH3 (residues at EU positions 341-447 of human IgG1).

Fc can refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein. Polymorphisms have been observed at a number of positions in Fc domains, including but not limited to EU positions 270, 272, 312, 315, 356, and 358. Thus, a “wild type IgG Fc domain” or “WT IgG Fc domain” refers to any naturally occurring IgG Fc region (i.e., any allele). Myriad Fc mutants, Fc fragments, Fc variants, and Fc derivatives are described, e.g., in U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056; 7,122,637; 7,183,387; 7,332,581; 7,335,742; 7,371,826; 6,821,505; 6,180,377; 7,317,091; 7,355,008; U.S. Patent publication 2004/0002587; and PCT Publication Nos. WO 99/058572, WO 2011/069164 and WO 2012/006635.

The sequences of the heavy chains of human IgG1, IgG2, IgG3 and IgG4 can be found in a number of sequence databases, for example, at the Uniprot database (www.uniprot.org) under accession numbers P01857 (IGHG1_HUMAN), P01859 (IGHG2_HUMAN), P01860 (IGHG3_HUMAN), and P01861 (IGHG1_HUMAN), respectively.

The terms “YTE” or “YTE mutant” refer to a set of mutations in an IgG1 Fc domain that results in an increase in the binding to human FcRn and improves the serum half-life of the antibody having the mutation. A YTE mutant comprises a combination of three “YTE mutations”: M252Y, S254T, and T256E, wherein the numbering is according to the EU index as in Kabat, introduced into the heavy chain of an IgG. See U.S. Pat. No. 7,658,921, which is incorporated by reference herein. The YTE mutant has been shown to increase the serum half-life of antibodies compared to wild-type versions of the same antibody. See, e.g., Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006) and U.S. Pat. No. 7,083,784, which are hereby incorporated by reference in their entireties. A “Y” mutant comprises only the M256Y mutations; similarly a “YT” mutation comprises only the M252Y and S254T; and a “YE” mutation comprises only the M252Y and T256E. It is specifically contemplated that other mutations may be present at EU positions 252 and/or 256. In certain aspects, the mutation at EU position 252 may be M252F, M252S, M252W or M252T and/or the mutation at EU position 256 may be T256S, T256R, T256Q or T256D.

The term “N3” or “N3 mutant” refers to a set of mutations in an IgG1 Fc domain that results in an increase in the binding to FcRn and improves the serum half-life of the antibody having the mutation. The N3 mutant comprises the sequence Cys-Ser-Trp-His-Leu-Cys at positions 432-437 (no insertion between positions 437 and 438), incorporated into a wild type IgG1 constant domain base structure. See WO2015175874, which is hereby incorporated by reference.

The term “naturally occurring O2” generally refers to a state in which the O2 polysaccharide or a fragment thereof can occur. Naturally occurring O2 means O2 polysaccharide which is naturally produced by a cell, without prior introduction of encoding nucleic acid using recombinant technology. Thus, naturally occurring O2 can be as produced naturally by for example K. pneumoniae and/or as isolated from different members of the Klebsiella genus.

The term “recombinant O2” refers to a state in which the O2 polysaccharide or fragments thereof may occur. Recombinant O2 means O2 polysaccharide or fragments thereof produced by recombinant DNA, e.g., in a heterologous host.

Recombinant proteins expressed in prokaryotic bacterial expression systems are not glycosylated while those expressed in eukaryotic systems such as mammalian or insect cells are glycosylated. Proteins expressed in insect cells however differ in glycosylation from proteins expressed in mammalian cells.

The terms “half-life” or “in vivo half-life” as used herein refer to the biological half-life of a particular type of antibody, antigen binding protein, or polypeptide of the present disclosure in the circulation of a given animal and is represented by a time required for half the quantity administered in the animal to be cleared from the circulation and/or other tissues in the animal.

The term “subject” as used herein refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, sheep, dogs, cats, horses, cows, bears, chickens, amphibians, reptiles, and the like, which is to be the recipient of a particular treatment. The terms “subject” and “patient” as used herein refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy of a condition associated with a Klebsiella infection. As used herein, phrases such as “a patient having a condition associated with a Klebsiella infection” includes subjects, such as mammalian subjects, that would benefit from the administration of a therapy, imaging or other diagnostic procedure, and/or preventive treatment for that condition associated with a Klebsiella infection.

“Klebsiella” refers to a genus of gram-negative, facultatively anaerobic, rod-shaped bacteria in the Enterobacteriaceae family. Klebsiella include, for example, K. pneumoniae, K oxytoca, K. planticola K. granulomatis, K. ozaenae, and K. rhinosclermoatis.

Members of the Klebsiella genus typically express 2 types of antigens on their cell surface: an O antigen and a K antigen. The O antigen is a lipopolysaccharide, and the K antigen is a capsular polysaccharide. The structural variability of these antigens forms the basis for their classification in into Klebsiella “serotypes.” Thus, the ability of an O2 binding protein (e.g., an antibody or an antigen binding fragment thereof) to bind to multiple serotypes refers to its ability to bind to Klebsiella with different 0 and/or K antigens. In some embodiments, provided herein, the Klebsiella is of the O2 serotype. In some embodiments, provided herein, the Klebsiella is of the O1 serotype.

The term “pharmaceutical composition” as used herein refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

An “effective amount” of an antigen binding protein (including an antibody or antigen binding fragment thereof), as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose. The term “therapeutically effective amount” as used herein refers to an amount of a polypeptide, e.g., an antigen binding protein including an antibody, or other drug effective to “treat” a disease or condition in a subject or mammal and provides some improvement or benefit to a subject having a Klebsiella-mediated disease or condition. Thus, a “therapeutically effective” amount is an amount that provides some alleviation, mitigation, and/or decrease in at least one clinical symptom of the Klebsiella-mediated disease or condition. Clinical symptoms associated with the Klebsiella-mediated disease or condition that can be treated by the methods and systems of the disclosure are well known to those skilled in the art. Further, those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. In some embodiments, the term “therapeutically effective” refers to an amount of a therapeutic agent that is capable of reducing Klebsiella (e.g., K. pneumoniae) or Klebsiella (e.g., K. pneumoniae) activity in a patient in need thereof. The actual amount administered and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibodies and antigen binding fragments thereof are well known in the art; see Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe K. D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922.

As used herein, a “sufficient amount” or “an amount sufficient to” achieve a particular result in a patient having a Klebsiella-mediated disease or condition refers to an amount of a therapeutic agent (e.g., an antigen binding protein including an antibody, as disclosed herein) that is effective to produce a desired effect, which is optionally a therapeutic effect (i.e., by administration of a therapeutically effective amount). In some embodiments, such particular result is a reduction in Klebsiella (e.g., K. pneumoniae) or Klebsiella (e.g., K. pneumoniae) activity in a patient in need thereof.

The term “label” when used herein refers to a detectable compound or composition which is conjugated directly or indirectly to a polypeptide, e.g., an antigen binding protein including an antibody, so as to generate a “labeled” polypeptide or antibody. The label can be detectable by itself (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, can catalyze chemical alteration of a substrate compound or composition which is detectable.

Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” or “ameliorating” or “or ameliorate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder. Terms such as “preventing” refer to prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disease or condition. Those in need of prevention include those prone to have the disease or condition and those in whom the disease or condition is to be prevented. For example, the phrase “treating a patient having a Klebsiella-mediated disease or condition” refers to reducing the severity of the Klebsiella-mediated disease or condition, preferably, to an extent that the subject no longer suffers discomfort and/or altered function due to it (for example, a relative reduction in asthma exacerbations when compared to untreated patients). The phrase “preventing a Klebsiella-mediated disease or condition” refers to reducing the potential for a Klebsiella-mediated disease or condition and/or reducing the occurrence of the Klebsiella-mediated disease or condition.

As used herein, the term “a condition associated with a Klebsiella infection” refers to any pathology caused by (alone or in association with other mediators), exacerbated by, associated with, or prolonged by Klebsiella infection (e.g. infection with K. pneumoniae, K. oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis and/or K. granulomatis) in the subject having the disease or condition. Non-limiting examples of conditions associated with a Klebsiella infection include pneumonia, urinary tract infection, septicemia/sepsis, neonatal septicemia, diarrhea, soft tissue infections, infections following an organ transplant, surgery infection, wound infection, lung infection, pyogenic liver abscesses, endophthalmitis, meningitis, necrotizing meningitis, ankylosing spondylitis and spondyloarthropathies. In some embodiments, the Klebsiella infection is a nosocomial infection. In some embodiments, the Klebsiella infection is an opportunistic infection. In some embodiments, the Klebsiella infection follows an organ transplant. In some embodiments, the subject is exposed to a Klebsiella contaminated medical device, including, e.g., a ventilator, a catheter, or an intravenous catheter.

The structure for carrying a CDR or a set of CDRs will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to Kabat, E. A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. (US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu or find “Kabat” using any search engine), herein incorporated by reference. CDRs can also be carried by other scaffolds such as fibronectin or cytochrome B.

A CDR amino acid sequence substantially as set out herein can be carried as a CDR in a human variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set out herein represent embodiments of the present disclosure and each of these may be carried as a HCDR3 in a human heavy chain variable domain or a substantial portion thereof.

Variable domains employed in the disclosure can be obtained from any germ-line or rearranged human variable domain, or can be a synthetic variable domain based on consensus sequences of known human variable domains. A CDR sequence (e.g. CDR3) can be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology.

For example, Marks et al. (Bio/Technology, 1992, 10:779-783; which is incorporated herein by reference) provide methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5′ end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3. Marks et al. further describe how this repertoire can be combined with a CDR3 of a particular antibody. Using analogous techniques, the CDR3-derived sequences of the present disclosure can be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide antigen binding proteins. The repertoire can then be displayed in a suitable host system such as the phage display system of WO92/01047 or any of a subsequent large body of literature, including Kay, B. K., Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press, so that suitable antigen binding proteins may be selected. A repertoire can consist of from anything from 104 individual members upwards, for example from 106 to 108 or 1010 members. Other suitable host systems include yeast display, bacterial display, T7 display, ribosome display and so on. For a review of ribosome display for see Lowe D and Jermutus L, 2004, Curr. Pharm, Biotech, 517-27, also WO92/01047, which are herein incorporated by reference.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer (Nature, 1994, 370:389-391, which is herein incorporated by reference), who describes the technique in relation to a β-lactamase gene but observes that the approach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying CDR-derived sequences of the disclosure using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR. In some embodiments, one or two amino acid substitutions are made within a set of HCDRs and/or LCDRs.

Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).

The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), all of which are herein incorporated by reference.

The skilled person will be able to use such techniques described above to provide antigen binding proteins of the present disclosure using routine methodology in the art.

II. O2 Antigen Binding Molecules

The present disclosure provides O2 antigen binding molecules, e.g., antigen binding proteins, antibodies, and antigen binding fragments thereof, that specifically bind K. pneumoniae O2 antigen. Collectively, these agents are referred to herein as “O2 binding molecules” or “O2 binding agents.” In some instances, an O2 binding agent (e.g., an antibody or antigen-binding fragment thereof) binds to K. pneumoniae O1 antigen in addition to binding to K. pneumoniae O2 antigen. In some instances, an O2 binding agent (e.g., an antibody or antigen-binding fragment thereof) binds to K. pneumoniae O1 antigen, but binds preferentially to K. pneumoniae O2 antigen. In some instances, an O2 binding agent (e.g., an antibody or antigen-binding fragment thereof) binds to K. pneumoniae O2 antigen but does not bind K. pneumoniae O1 antigen.

The O2 antigen of Klebsiella lipopolysaccharide (LPS) contains repeating D-galactan I (D-Gal I) units as major structural components. In the O2 antigen, the D-Gal I polymers are directly linked to the core oligosaccharide and are composed of repeat units of the structure→3)-β-D-Galf-(1→3)-α-D-Galp-(1→. In contrast, the O1 antigen of Klebsiella lipopolysaccharide (LPS) contains two structural domains composed of the repeat units D-galactan I and D-galactan II. For both the O1 antigen and the O2 antigen of Klebsiella, O-antigen biosynthesis is performed by products of the wb (rfb) gene cluster, which is composed of six genes (wzm, wzt, glf, wbbM, wbbN, and wbbO) (Whitfield, C. et al. 1991. Expression of two structurally distinct D-Galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O2. J. Bacteriology. 1420-1431; Clarke, B. R. and Whitfield C. 1992. Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O2:K20. J. Bacteriology. 174: 4614-4621). D-Gal I domain is also the major 0-antigen component for Klebsiella O2 LPS. See FIG. 1A.

Several O2 sub-serotypes have been discussed in the literature. (Kelly, R. F., et al. 1996. Clonally diverse rfb gene clusters are involved in expression of a family of related D-Galactan O antigens in Klebsiella species. J. Bacteriology. 5205-5214.) Structural analyses showed that sub-serotype O2 (2a) expresses only the D-Gal I moiety, while other sub-serotypes, such as O2 (2a, 2c), O2 (2a, 2e), O2 (2a, 2e, 2 h), etc. contain additional modifications on the D-Gal I backbone. Recently, →3)-β-D-Galf-(1→3)-[α-D-Galp-(1→4)]-α-D-Galp-(1→trisaccharide repeating unit of sub-serotype O2 (2a, 2f, 2g) were defined as D-Galactan III domain that was expressed in a subset of highly resistant CRE strains (clonal complex 258). The Galactan I modifying locus (gml) gene was responsible for the Galactan III domain, which has an additional galactose attached to D-galactopyranose on D-Gal I repeating units. (Szijarto, V. et. al. Int J Med Microbiol. 306(2):89-98 (2016)).

As used herein, the phrase “O2 antigen” includes both gml+ and gml− O2 LPS and does not include the D-galactose side chain of D-galactan III (D-Gal III) units. Accordingly, an “O2 binding agent” (e.g., an antibody or antigen-binding fragment thereof) as defined herein does not bind specifically to D-Gal III. Thus, in some embodiments, an O2 binding agent (e.g., an antibody or antigen-binding fragment thereof) can bind to an O2 antigen regardless of D-Gal III expression (Table 8). In some embodiments, an O2 binding agent (e.g., an antibody or antigen-binding fragment thereof) can bind to an O2 antigen that contains both D-Gal I and D-Gal III, e.g., by binding to the common carbohydrate moieties expressed in all O2 strains.

In some embodiments, the disclosure provides an isolated antigen binding protein that is an antibody or polypeptide that specifically binds to K. pneumoniae O2 antigen. In some embodiments, the antigen binding protein is an antigen binding fragment of an antibody that specifically binds to K. pneumoniae O2 antigen.

In certain embodiments, the O2 binding molecules are antibodies or polypeptides. In some embodiments, the disclosure provides an isolated antigen binding protein that is a murine, non-human, humanized, chimeric, resurfaced, or human antigen binding protein that specifically binds to K. pneumoniae O2 antigen. In some embodiments, the O2 binding molecules are humanized antibodies or antigen binding fragment thereof. In some embodiments, the O2 binding molecule is a human antibody or antigen binding fragment thereof.

The disclosure provides an isolated antigen binding protein (including an antibody or antigen binding fragment thereof) that specifically binds to K. pneumoniae O2 antigen, wherein said antigen binding protein (e.g., an antibody or antigen binding fragment thereof): a) induces opsonophagocytic killing (OPK) of a Klebsiella, b) protects mice from a lethal Klebsiella challenge or c) induces OPK of a Klebsiella and protects mice from a lethal Klebsiella challenge. In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) does not have LPS neutralization activity (e.g., as determined using the assay described in Example 7). In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) has LPS neutralization activity against O2 LPS (e.g., as determined using the assay described in Example 7). In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) has LPS neutralization activity against both O1 and O2 LPS (e.g., as determined using the assay described in Example 7). In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) does not inhibit, reduce, or prevent NF-kB activation induced by LPS. In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) inhibits, reduces, or prevents NF-kB activation induced by O2 LPS. In certain embodiments, the isolated antigen binding protein (including an antibody or antigen binding fragment thereof) inhibits, reduces, or prevents NF-kB activation induced by both O1 and O2 LPS.

The O2-binding agents include anti-O2 antigen antibodies KPN42, KPN42-FR-1-2-4-GL-VH/KPN42-FR-GL-C105A-VL (KPN42-v2016), KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, KPD1, and antigen-binding fragments thereof. The O2-binding agents also include O2-binding agents (e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) that specifically bind to the same K. pneumoniae O2 epitope as KPN42, KPN42-FR-1-2-4-GL-VH/KPN42-FR-GL-C105A-VL, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1. In some embodiments, the O2-binding agents disclosed herein include anti-O2 antigen antibodies or antigen binding fragments thereof that bind to the D-Galactan I domain of K. pneumoniae O2 antigen.

The O2-binding agents (e.g. anti-O2 antigen antibodies or antigen binding fragments thereof) also include O2-binding agents that competitively inhibit binding of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 to K. pneumoniae O2 antigen. In some embodiments, an anti-O2 antibody or antigen-binding fragment thereof competitively inhibits binding of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 to K. pneumoniae O2 antigen in a competition ELISA assay. In some embodiments, an anti-O2 antibody or antigen-binding fragment thereof competitively inhibits binding of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 to K. pneumoniae in a competition ELISA assay.

The O2-binding agents (e.g. anti-O2 antigen antibodies or antigen binding fragments thereof) also include O2-binding agents that bind Klebsiella of the O2 serotype, independent of gml gene expression. The effect of gml gene expression can be assessed, for example, using the methods provided in Szijártó et al., International Journal of Medical Microbiology 306: 89-98 (2016), which is herein incorporated by reference in its entirety. In some embodiments, the O2-binding agents disclosed herein include anti-O2 antigen antibodies or antigen binding fragments thereof that bind Klebsiella of the O2 serotype that do not express the gml gene (i.e., gml− Klebsiella). In some embodiments, the O2-binding agents disclosed herein include anti-O2 antigen antibodies or antigen binding fragments thereof that bind Klebsiella of the O2 serotype that express the gml gene (i.e., gml+Klebsiella).

The O2-binding agents (e.g. anti-O2 antigen antibodies or antigen binding fragments thereof) also include O2-binding agents that comprise the heavy and light chain complementarity determining region (CDR) sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1. The CDR sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS30, and KPD1 are described in Tables 1 and 2 below.

TABLE 1

Variable heavy chain CDR amino acid sequences

Antibody
VH-CDR1
VH-CDR2
VH-CDR3

KPN42
GFTFNDAW (SEQ ID
IKKKHEGVTT (SEQ ID
TTRIVTTNDY (SEQ ID

NO: 1)
NO: 2)
NO: 3)

KPN42--
GFTFNDAW (SEQ ID
IKKKHEGVTT (SEQ ID
TTRIVTTNDY (SEQ ID

FR-1-2-4-
NO: 10)
NO: 11)
NO: 12)

GL-

VH/KPN42-

FR-GL-

C105A-VL

(KPN42-

v2016)

KPN42-
GFTFNDAW (SEQ ID
IKKKHEGVTT (SEQ ID
TTRIVTTNDY (SEQ ID

FR-GL-
NO: 19)
NO: 20)
NO: 21)

VH/KPN42-

FR-GL-

C105A-VL

KPS3
GFSFRDYG (SEQ ID
ISYDGRDQ (SEQ ID
GPFYNPSLYYPP (SEQ ID

NO: 28)
NO: 29)
NO: 30)

KPN70
GGSISTYY (SEQ ID
IHQSGTT (SEQ ID NO: 38)
ARESDDGYKWNYFDY

NO: 37)

(SEQ ID NO: 39)

KPN179
GFTFNNAW (SEQ ID
IKRKADGETT (SEQ ID
TTRIVTTNDY (SEQ ID

NO: 46)
NO: 47)
NO: 48)

KPN179-
GFTFSNAW (SEQ ID
IKRKADGETT (SEQ ID
TTRIVTTNDY (SEQ ID

FR-1-2-4-
NO: 166)
NO: 167)
NO: 168)

GL-N35S-

VH/KPN179-

FR-GL-

C105A-VL

KPN179-
GFTFSNAW (SEQ ID
IKRKADGETT (SEQ ID
TTRIVTTNDY (SEQ ID

FR-GL
NO: 169)
NO: 170)
NO: 171)

N35S-

VH/KPN179-

FR-GL-

C105A-VL

KPN44
GGSTSSYY (SEQ ID
IHHGGTT (SEQ ID NO: 56)
ARESDDGYRWNYFDY

NO: 55)

(SEQ ID NO: 57)

KPN17
GFTFSHFW (SEQ ID
IDGSVTNL (SEQ ID NO: 65)
ARDLVGIGTPAGYGMD

NO: 64)

V (SEQ ID NO: 66)

6F6
PIAYMG (SEQ ID
DILPNIGRTIYGEKFED
RGTSGAMDY (SEQ ID

NO: 73)
(SEQ ID NO: 74)
NO: 75)

KPL26
GFIFGSSW (SEQ ID
INPDGSAT (SEQ ID NO: 83)
TRNKAYNALDY (SEQ ID

NO: 82)

NO: 84)

KPS18
GFTFKNAW (SEQ ID
VKNEVDGGTI (SEQ ID
RAFWSGFPAGY (SEQ ID

NO: 91)
NO: 92)
NO: 93)

KPS24
GFTFKNAW (SEQ ID
VKSEVDGGTT (SEQ ID
RAFWSDFQTGY (SEQ ID

NO: 100)
NO: 101)
NO: 102)

KPS44
GFTFKNAW (SEQ ID
VKSEVDGGTI (SEQ ID
RAFWSGFPTGY (SEQ ID

NO: 109)
NO: 110)
NO: 111)

KPS44-
GFTFKNAW (SEQ ID
VKSEVDGGTI (SEQ ID
RAFFSGFPTGY (SEQ ID

v2017
NO: 109)
NO: 110)
NO: 199)

KPS44-G1
GFTFKNAW (SEQ ID
VKSEVDGGTI (SEQ ID
RAFWSGFPTGY (SEQ ID

NO: 109)
NO: 110)
NO: 111)

KPS44-G2
GFTFKNAW (SEQ ID
VKSEVDGGTI (SEQ ID
RAFFSGFPTGY (SEQ ID

NO: 109)
NO: 110)
NO: 199)

KPS44-G3
GFTFKNAW (SEQ ID
VKSEVDGGTI (SEQ ID
RAFFSGFPTGY (SEQ ID

NO: 109)
NO: 110)
NO: 199)

KPS30
GFSFSTSW (SEQ ID
IDPDGSTR (SEQ ID
ARDYAYNRFDY (SEQ ID

NO: 118)
NO: 119)
NO: 120)

KPD1
GVSITSNTYW (SEQ ID
LSYSGDT (SEQ ID NO: 128)
ARDPDIIRNFQFDY (SEQ

NO: 127)

ID NO: 129)

KPL36
GFTFISSW (SEQ ID
INPDGTET (SEQ ID
ARNKAYNAHDF (SEQ ID

NO: 172)
NO: 173)
NO: 174)

KPS44-G4
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

KPS44-G6
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

KPS44-G8
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

KPS44-G10
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

KPS44-G11
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

KPS44-G14
GFTFKNAW (SEQ ID
VKSEVDAGTI (SEQ ID
RAFYSGFPTGY (SEQ ID

NO: 109)
NO: 215)
NO: 216)

TABLE 2

Variable light chain CDR amino acid sequences

Antibody
VL-CDR1
VL-CDR2
VL-CDR3

KPN42
SSDVGAYDY (SEQ ID
DVN (SEQ ID NO: 5) or
CSYAGGDIFV (SEQ ID

NO: 4)
IIYDVNERP (SEQ ID
NO: 7)

NO: 6)

KPN42--
SSDVGAYDY (SEQ ID
DVN (SEQ ID NO: 14) or
ASYAGGDIFV (SEQ ID

FR-1-2-4-
NO: 13)
MIYDVNKRP (SEQ ID
NO: 16)

GL-

NO: 15)

VH/KPN42-

FR-GL-

C105A-VL

(KPN42-

v2016)

KPN42-
SSDVGAYDY (SEQ ID
DVN (SEQ ID NO: 23) or
ASYAGGDIFV (SEQ ID

FR-GL-
NO: 22)
MIYDVNKRP (SEQ ID
NO: 25)

VH/KPN42-

NO: 24)

FR-GL-

C105A-VL

KPS3
QSISSQ (SEQ ID NO: 31)
DAS (SEQ ID NO: 32) or
LQRNNWPPWT (SEQ ID

LIHDASNRD (SEQ ID
NO: 34)

NO: 33)

KPN70
QIVTNY (SEQ ID NO: 40)
DMS (SEQ ID NO: 41) or
QHRSNWPLFT (SEQ ID

LIFDMSIRA (SEQ ID
NO: 43)

NO: 42)

KPN179
SSDVGYYDY (SEQ ID
DVN (SEQ ID NO: 50) or
CSYAGGDTFV (SEQ ID

NO: 49)
MIYDVNKRP (SEQ ID
NO: 52)

NO: 51)

KPN179-
SSDVGYYDY (SEQ ID
DVN (SEQ ID NO: 176) or
ASYAGGDTFV (SEQ ID

FR-1-2-4-
NO: 175)
MIYDVNKRP (SEQ ID
NO: 178)

GL-N35S-

NO: 177)

VH/KPN179-

FR-GL-

C105A-VL

KPN179-
SSDVGYYDY (SEQ ID
DVN (SEQ ID NO: 180) or
ASYAGGDTFV (SEQ ID

FR-GL
NO: 179)
MIYDVNKRP (SEQ ID
NO: 182)

N35S-

NO: 181)

VH/KPN179-

FR-GL-

C105A-VL

KPN44
QTITNY (SEQ ID NO: 58)
DMS (SEQ ID NO: 59) or
QHRSNWPLFT (SEQ ID

LIFDMSKRA (SEQ ID
NO: 61)

NO: 60)

KPN17
QGISTY (SEQ ID NO: 67)
AAS (SEQ ID NO: 68) or
QQLTSHLYT (SEQ ID

LIYAASTLQ (SEQ ID
NO: 70)

NO: 69)

6F6
RSSQGLVHSTGNTFLH
KVSNRFS (SEQ ID NO:
SQSTHIPYT (SEQ ID NO:

(SEQ ID NO: 76)
77)
78)

KPL26
SSDVGGNNY (SEQ ID
EVS (SEQ ID NO: 86) or
SSFGGSKM (SEQ ID

NO: 85)
IIYEVSKRP (SEQ ID
NO: 88)

NO: 87)

KPS18
RSNIGSDS (SEQ ID
DNN (SEQ ID NO: 95) or
ATWDSSLSAYV (SEQ ID

NO: 94)
LMYDNNKRP (SEQ ID
NO: 97)

NO: 96)

KPS24
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 104) or
AAWDSSLRAYV (SEQ ID

NO: 103)
LMYENNKRP (SEQ ID
NO: 106)

NO: 105)

KPS44
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATWDSSLSAYV (SEQ ID

NO: 112)
LIYENNKRP (SEQ ID
NO: 115)

NO: 114)

KPS44-
SSNIGSNY (SEQ ID
ENN (SEQ ID NO: 113) or
ATFDSSLSAYV (SEQ ID

v2017
NO: 200)
LIYENNKRP (SEQ ID
NO: 201)

NO: 114)

KPS44-G1
SSNIGSNY (SEQ ID
ENN (SEQ ID NO: 113) or
ATFDSSLSAYV (SEQ ID

NO: 200)
LIYENNKRP (SEQ ID
NO: 201)

NO: 114)

KPS44-G2
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATFDSSLSAYV (SEQ ID

NO: 112)
LIYENNKRP (SEQ ID
NO: 201)

NO: 114)

KPS44-G3
SSNIGSNY (SEQ ID
ENN (SEQ ID NO: 113) or
ATWDSSLSAYV (SEQ ID

NO: 200)
LIYENNKRP (SEQ ID
NO: 115)

NO: 114)

KPS30
SSDIGANNY (SEQ ID
EVN (SEQ ID NO: 122) or
CGYGGGRV (SEQ ID

NO: 121)
LLYEVNKRP (SEQ ID
NO: 124)

NO: 123)

KPD1
QILYMSH (SEQ ID
GAS (SEQ ID NO: 131) or
QQYGASPT (SEQ ID

NO: 130)
LIYGASIRA (SEQ ID
NO: 133)

NO: 132)

KPL36
SSDVGGNNF (SEQ ID
EVN (SEQ ID NO: 184) or
GAFGGSKM (SEQ ID

NO: 183)
IIYEVNKRP (SEQ ID
NO: 186)

NO: 185)

KPS44-G4
SSNIGSDA (SEQ ID NO:
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

218)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

KPS44-G6
SSNIGSES (SEQ ID NO:
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

228)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

KPS44-G8
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

NO: 238)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

KPS44-G10
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

NO: 238)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

KPS44-G11
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

NO: 238)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

KPS44-G14
SSNIGSDS (SEQ ID
ENN (SEQ ID NO: 113) or
ATFESSLSAYV (SEQ

NO: 238)
LIYENNKRP (SEQ ID
ID NO: 230)

NO: 114)

Antigen binding proteins (including anti-O2 antigen antibodies or antigen binding fragments thereof) described herein can comprise one of the individual variable light chains or variable heavy chains described herein. Antigen binding proteins (including anti-O2 antigen antibodies or antigen binding fragments thereof) described herein can also comprise both a variable light chain and a variable heavy chain. The variable light chain and variable heavy chain sequences of anti-O2 antigen KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, and KPD1 antibodies are provided in Tables 3 and 4 below.

TABLE 3

Variable heavy chain amino acid sequences

Antibody
VH Amino Acid Sequence (SEQ ID NO)

KPN42
EVQLVESGGGLVKPGGSLRLSCAASGFTFNDAWMNWVRQAPGKGLEWVARIKK

KHEGVTTDYPASVRGRFTISRDDSKNTVYLQMGRLRIEDTAIYYCTTRIVTTNDYW

GQGTLVTVSS (SEQ ID NO: 8)

KPN42-
EVQLVESGGGLVKPGGSLRLSCAASGFTFNDAWMNWVRQAPGKGLEWVGRIKK

v2016
KHEGVTTDYPASVRGRFTISRDDSKNTVYLQMGRLRIEDTAIYYCTTRIVTTNDYW

GQGTLVTVSS (SEQ ID NO: 17)

KPN42-
EVQLVESGGGLVKPGGSLRLSCAASGFTFNDAWMNWVRQAPGKGLEWVGRIKK

FR-GL-
KHEGVTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTRIVTTNDY

VH/KPN42-
WGQGTLVTVSS (SEQ ID NO: 26)

FR-GL-

C105A-

VL

KPS3
QGQLVDSGGGVVQRGGSQRLSCAASGFSFRDYGMHWVRQAPGKGLEWVAFISY

DGRDQYYADSVKGRFIISRDNSKNTLSLQMNSLRPEDTAVYYCGPFYNPSLYYPP

WGHGLPVIVSS (SEQ ID NO: 35)

KPN70
QVQLQESGPGLVKPSETLSLTCTVSGGSISTYYWNWIRQSPGKELEWIANIHQSGTT

YYNPSLKSRVTMSVDTSKNQFSLKVISVTAADTAVYYCARESDDGYKWNYFDYW

GQGTLVTVSS (SEQ ID NO: 44)

KPN179
EVQVVESGGGLVKPGGSLRLSCAASGFTFNNAWMNWVRQAPGKGLEWVGRIKR

KADGETTDYPASVKGRFTVSRDDSKNTIYLQMNSLKTEDTAIYYCTTRIVTTNDY

WGQGTLVTVSS (SEQ ID NO: 53)

KPN179-
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMNWVRQAPGKGLEWVGRIKRK

FR-1-2-
ADGETTDYPASVKGRFTVSRDDSKNTIYLQMNSLKTEDTAIYYCTTRIVTTNDYW

4-GL-
GQGTLVTVSS (SEQ ID NO: 187)

N35S-

VH/KPN179-

FR-GL-

C105A-VL

KPN179-
EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMNWVRQAPGKGLEWVGRIKRK

FR-GL
ADGETTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTRIVTTNDYW

N35S -
GQGTLVTVSS (SEQ ID NO: 188)

VH/KPN

179-FR-

GL-

C105A-

VL

KPN44
QVQLQESGPGLVKPSETLSLTCTVSGGSTSSYYWNWIRQAPGKPLQWIANIHHGGT

TYYNPSLRSRVTMSLDTSNNQFSLKLTSVTAADTAVYFCARESDDGYRWNYFDY

WGQGVLVTVSS (SEQ ID NO: 62)

KPN17
EVQLVESGGGLVQPGGSLRLSCAASGFTFSHFWMHWVRQAPGQGLVWVARIDGS

VTNLRYAGSVEGRFTISRDNAKNTLYLQMNSLRDEDTAVYYCARDLVGIGTPAGY

GMDVWGQGTTVTVSS (SEQ ID NO: 71)

6F6
QVHLQQSGSELRSPGSSVKLSCKDFDSDVFPIAYMGWIRQQPGHGFDWIGDILPNI

GRTIYGEKFEDKATLDADTVSNTAYLELSSLTSEDSAIYYCARRGTSGAMDYWGQ

GTSVTVSS (SEQ ID NO: 80)

KPL26
EVQLVESGGGLVQSGGSLRLSCETSGFIFGSSWMTWVRQAPGKGLEWVATINPDG

SATSYEDSVRGRFAVSRDNAKNSVYLQMNSLRAEDTAVYFCTRNKAYNALDYW

GQGTLVTVSS (SEQ ID NO: 89)

KPS18
EVRLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKNE

VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLREDDTGIYYCRAFWSGFPAGY

WGQGTLVSVSS (SEQ ID NO: 98)

KPS24
ELHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDGGTTDYGVPVRGRFTISRDDSQSTLSLEMSSLQDDDTGVYYCRAFWSDFQTGY

WGQGTLVTVSS (SEQ ID NO: 107)

KPS44
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFWSGFPTGY

WGQGALVSVSS (SEQ ID NO: 116)

KPS44-
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

v2017
VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFFSGFPTGY

WGQGALVSVSS (SEQ ID NO: 202)

KPS44-G1
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFWSGFPTGY

WGQGALVSVSS (SEQ ID NO: 116)

KPS44-G2
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFFSGFPTGY

WGQGALVSVSS (SEQ ID NO: 202)

KPS44-G3
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDGGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFFSGFPTGY

WGQGALVSVSS (SEQ ID NO: 202)

KPS30
EMQLVESGGGLVQPGVSLRLSCVDSGFSFSTSWLAWVRQAPGKGLEWLANIDPD

GSTRNHVDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDYAYNRFDYW

GQGTMVTVSS (SEQ ID NO: 125)

KPD1
QVQLQESDPRLVKPSETLSLTCSVSGVSITSNTYWWAWIRQPPGKKLEWIGSLSYS

GDTYYNPSLTSRVTISRDIHQNQFFLELNSVTAADTAMYYCARDPDIIRNFQFDYW

GRGTLVTVSS (SEQ ID NO: 134)

KPL36
EVQLVESGGGVVQSGGSLRLSCETSGFTFISSWMSWVRQAPGTGLEWVATINPDG

TETPYADSLKGRFTISRDNTKKSLYLQIHSLRADDTAVYFCARNKAYNAHDFWGQ

GTLVMVSS (SEQ ID NO: 189)

KPS44-G4
QVQLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDAGTIDYGVPVRGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCRAFYSGFPTGY

WGQGTLVTVSS (SEQ ID NO: 213)

KPS44-G6
QVQLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDAGTIDYGVPVRGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCRAFYSGFPTGY

WGQGTLVTVSS (SEQ ID NO: 223)

KPS44-G8
QVQLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

VDAGTIDYGVPVRGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCRAFYSGFPTGY

WGQGTLVTVSS (SEQ ID NO: 233)

KPS44-
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

G10
VDAGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFYSGFPTGY

WGQGALVSVSS (SEQ ID NO: 243)

KPS44-
EVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

G11
VDAGTIDYGVPVRGRFTISRDDSQGTLSLEMNSLKDDDTGVYYCRAFYSGFPTGY

WGQGALVSVSS (SEQ ID NO: 253)

KPS44-
QVQLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

G14
VDAGTIDYGVPVRGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCRAFYSGFPTGY

WGQGTLVTVSS (SEQ ID NO: 263)

KPS44-
QVQLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

G8-
VDAGTIDYGVPVRGRFTISRDDSQGTLYLQMNSLKTEDTGVYYCRAFYSGFPTGY

HCvFW1
WGQGTLVTVSS (SEQ ID NO: 273)

KPS44-
QVHLVESGGGLVKPGGSLRLSCAASGFTFKNAWMSWIRQAPGKGLEWVGRVKSE

G8-
VDAGTIDYGVPVRGRFTISRDDSQGTLYLQMNSLKTEDTGVYYCRAFYSGFPTGY

HCvFW2
WGQGTLVTVSS (SEQ ID NO: 274)

TABLE 4

Variable light chain amino acid sequences

Antibody
VL Amino Acid Sequence (SEQ ID NO)

KPN42
QSALTQPPSVSGSPGQSVTISCTGTSSDVGAYDYVSWYQQYAGKVPKHIIYDVNER

PSGVPDRFSGSKSGNTAALTISGLQAEDEADYYCCSYAGGDIFVFGTGTQVTVL

(SEQ ID NO: 9)

KPN42-
QSALTQPRSVSGSPGQSVTISCTGTSSDVGAYDYVSWYQQHPGKAPKLMIYDVNK

v2016
RPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCASYAGGDIFVFGTGTKVTVL

(SEQ ID NO: 18)

KPN42-
QSALTQPRSVSGSPGQSVTISCTGTSSDVGAYDYVSWYQQHPGKAPKLMIYDVNK

FR-GL-
RPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCASYAGGDIFVFGTGTKVTVL

VH/KPN42-
(SEQ ID NO: 27)

FR-GL-

C105A-

VL

KPS3
EVVLTQSPATLSLSPGERATLSCRASQSISSQLAWYQQKPGQAPRLLIHDASNRDTG

VPDRFSGSGSGTDFTLTISSLEPEDFAMYYCLQRNNWPPWTFGQGTKVEIK (SEQ

ID NO: 36)

KPN70
EIVLTQSPASLSLSPGERATLSCRASQIVTNYLAWYQHKPGQAPRLLIFDMSIRAAGI

PARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLFTFGPGTKVDIK (SEQ ID

NO: 45)

KPN179
QSALTQPPSVSGSPGQSVTISCTGTSSDVGYYDYVSWYQQHHPGKAPKHMIYDVN

KRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGGDTFVFGTGTKVTVL

v(SEQ ID NO: 54)

KPN179-
QSALTQPRSVSGSPGQSVTISCTGTSSDVGYYDYVSWYQQHPGKAPKLMIYDVNK

FR-1-2-
RPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCASYAGGDTFVFGTGTKVTVL

4-GL-
(SEQ ID NO: 190)

N35S-

VH/KPN179-

FR-GL-

C105A-

VL

KPN179-
QSALTQPRSVSGSPGQSVTISCTGTSSDVGYYDYVSWYQQHPGKAPKLMIYDVNK

FR-GL
RPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCASYAGGDTFVFGTGTKVTVL

N35S-
(SEQ ID NO: 191)

VH/KPN179-

FR-GL-

C105A-

VL

KPN44
EIVLTQSPASLSLSPGDRATLSCRASQTITNYLAWYQHKPGQAPRLLIFDMSKRATG

IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHRSNWPLFTFGPGTNVDIK (SEQ ID

NO: 63)

KPN17
DIQLTQSPSFLSASVGDRVTITCRASQGISTYLAWYQQKPGKAPKLLIYAASTLQSG

VPSRFSGSGSGTEFTLTINSLQSEDFATYYCQQLTSHLYTFGQGTKLEIK (SEQ ID

NO: 72)

6F6
DVVMTQTPLFLPVSLGDQASISCRSSQGLVHSTGNTFLHWYLQKPGQSPKLLIYKV

SNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYFCSQSTHIPYTFGGGTKLEIK

(SEQ ID NO: 81)

KPL26
QSALTQPPSASGSPGQSVTLSCTGTSSDVGGNNYVSWYQQHPGKAPKLIIYEVSKR

PSGVPNRFSGSKSGNTASLTVSGLQAEDEADYYCSSFGGSKMFGGGTKLTVL (SEQ

ID NO: 90)

KPS18
QSVLTQPPSLSAAPGQTVTIACSGSRSNIGSDSVSWFQQFPGTAPRVLMYDNNKRP

SGISDRFSGSKSGTSVTLDITGLQTGDEADYYCATWDSSLSAYVFGSGTKVTVL

(SEQ ID NO: 99)

KPS24
QSVLTQPPSVSAAPGQTVTIACSGSSSNIGSDSVSWFQQLPGTAPRVLMYENNKRPS

GISDRFSGSKSGTSVTLGITGLQTGDEADYYCAAWDSSLRAYVFGSGTKVTVL

(SEQ ID NO: 108)

KPS44
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSDSVSWFQQFPGTAPRVLIYENNKRPSGI

SDRFSGSKSGTSVTLGITGLQTGDEADYYCATWDSSLSAYVFGSGTKVTVL (SEQ

ID NO: 117)

KPS44-
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSNYVSWFQQFPGTAPRVLIYENNKRPSG

v2017
ISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFDSSLSAYVFGSGTKVTVL (SEQ

ID NO: 203)

KPS44-G1
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSNYVSWFQQFPGTAPRVLIYENNKRPSG

ISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFDSSLSAYVFGSGTKVTVL (SEQ

ID NO: 203)

KPS44-G2
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSDSVSWFQQFPGTAPRVLIYENNKRPSGI

SDRFSGSKSGTSVTLGITGLQTGDEADYYCATFDSSLSAYVFGSGTKVTVL (SEQ

ID NO: 204)

KPS44-G3
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSNYVSWFQQFPGTAPRVLIYENNKRPSG

ISDRFSGSKSGTSVTLGITGLQTGDEADYYCATWDSSLSAYVFGSGTKVTVL (SEQ

ID NO: 205)

KPS30
QSALTQPPSASGSPGQSVVISCTGTSSDIGANNYVSWYQQHPGKAPKLLLYEVNKR

PSGVPDRFSASKSGNTASLTVSGLLAEDEADYYCCGYGGGRVFGGGTKLTVL

(SEQ ID NO: 126)

KPD1
EIVLTQSPGILSLSPGERATLSCRVSQILYMSHLAWYQHKPGQAPRLLIYGASIRAT

GVPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGASPTFGQGTMVEIK (SEQ ID

NO: 135)

KPL36
QSALTQPPSASGSPGQSVTISCTGTSSDVGGNNFVSWYQQYPGKAPKLIIYEVNKRP

SGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCGAFGGSKMFGGGTKLTVL (SEQ

ID NO: 192)

KPS44-G4
QSVLTQPPSVSAAPGQKVTIACSGTSSNIGSDAVSWFQQLPGTAPKLLIYENNKRPS

GISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGTGTKVTVL (SEQ

ID NO: 217)

KPS44-G6
QSVLTQPPSVSAAPGQKVTIACSGTSSNIGSESVSWFQQLPGTAPKLLIYENNKRPS

GISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGTGTKVTVL (SEQ

ID NO: 227)

KPS44-G8
QSVLTQPPSVSAAPGQKVTIACSGTSSNIGSDSVSWFQQLPGTAPKLLIYENNKRPS

GISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGTGTKVTVL (SEQ

ID NO: 237)

KPS44-
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSDSVSWFQQFPGTAPRVLIYENNKRPSGI

G10
SDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGSGTKVTVL (SEQ ID

NO: 247)

KPS44-
QSVLTQPPSVSAAPGQKVTIACSGTSSNIGSDSVSWFQQLPGTAPKLLIYENNKRPS

G11
GISDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGTGTKVTVL (SEQ

ID NO: 257)

KPS44-
QSVLTQPPSLSAAPGQTITIACSGTSSNIGSDSVSWFQQFPGTAPRVLIYENNKRPSGI

G14
SDRFSGSKSGTSVTLGITGLQTGDEADYYCATFESSLSAYVFGSGTKVTVL (SEQ ID

NO: 267)

In some embodiments, the disclosure provides an isolated antigen binding protein (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) that specifically binds to K. pneumoniae O2 antigen, wherein said antigen binding protein comprises a heavy chain variable region (VH) at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 8, 17, 26, 35, 44, 53, 187, 188, 62, 71, 80, 89, 98, 107, 116, 125, 134, or 189 and a light chain variable region (VL) at least 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 9, 18, 27, 36, 45, 54, 190, 191, 63, 72, 81, 90, 99, 108, 117, 126, 135, or 192. In some embodiments, the isolated antigen binding protein (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) that specifically binds to K. pneumoniae O2 antigen comprises a heavy chain variable region comprising the sequences of SEQ ID NO: 8, 17, 26, 35, 44, 53, 187, 188, 62, 71, 80, 89, 98, 107, 116, 125, 134, or 189 and a light chain variable region comprising the sequences of SEQ ID NOs: 9, 18, 27, 36, 45, 54, 190, 191, 63, 72, 81, 90, 99, 108, 117, 126, 135, or 192. In some embodiments, the isolated antigen binding protein (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) having least 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 8, 9, 17, 18, 26, 27, 35, 36, 44, 45, 53, 54, 187, 190, 188, 191, 62, 63, 71, 72, 80, 81, 89, 90, 98, 99, 107, 108, 116, 117, 125, 126, 134, 135, 189, or 192 differs from SEQ ID NO: 8, 9, 17, 18, 26, 27, 35, 36, 44, 45, 53, 54, 187, 190, 188, 191, 62, 63, 71, 72, 80, 81, 89, 90, 98, 99, 107, 108, 116, 117, 125, 126, 134, 135, 189, or 192 by conservative amino acid substitutions only.

Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein (1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies above.

Alternatively monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

In some embodiments, the monoclonal antibody against the K. pneumoniae O2 antigen is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject. Humanized antibodies can be produced using various techniques known in the art. In certain alternative embodiments, the antibody to K. pneumoniae O2 antigen is a human antibody.

Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Pat. No. 5,750,373). Also, the human antibody can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al., 1991, J. Mol. Biol., 222:581). Techniques for the generation and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., 2008, J. Mol. Bio., 376: 1182-200 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al., 1992, Bio/Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be employed to generate high affinity human antibodies.

Humanized antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

According to the present disclosure, techniques can be adapted for the production of single-chain antibodies specific to K. pneumoniae O2 antigen (see U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (Huse, et al., Science 246:1275-1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for O2 antigen, or fragments thereof. Antibody fragments can be produced by techniques in the art including, but not limited to: (a) a F(ab′)2 fragment produced by pepsin digestion of an antibody; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab′)2 fragment, (c) a Fab fragment generated by the treatment of the antibody with papain and a reducing agent, and (d) Fv fragments.

It can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).

Antigen binding proteins of the present disclosure (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) can further comprise antibody constant regions or parts thereof. For example, a VL domain can be attached at its C-terminal end to antibody light chain constant domains including human Cκ or Cγ chains. Similarly, an antigen binding protein based on a VH domain can be attached at its C-terminal end to all or part (e.g. a CH1 domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1 and IgG4. For example, the immunoglobulin heavy chain can be derived from the antibody isotype sub-class, IgG1. Any synthetic or other constant region variant that has these properties and stabilizes variable regions is also contemplated for use in embodiments of the present disclosure. The antibody constant region can be an Fc region with a YTE mutation, such that the Fc region comprises the following amino acid substitutions: M252Y/S254T/T256E. This residue numbering is based on Kabat numbering. The YTE mutation in the Fc region increases serum persistence of the antigen-binding protein (see Dall'Acqua, W. F. et al. (2006) The Journal of Biological Chemistry, 281, 23514-23524).

In some embodiments herein, the antigen binding protein, e.g., antibody or antigen-binding fragment thereof, is modified to improve effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC). This can be achieved by making one or more amino acid substitutions or by introducing cysteine in the Fc region. Variants of the Fc region (e.g., amino acid substitutions and/or additions and/or deletions) that can enhance or diminish effector function of an antibody and/or alter the pharmacokinetic properties (e.g., half-life) of the antibody are disclosed, for example in U.S. Pat. No. 6,737,056B1, U.S. Patent Application Publication No. 2004/0132101A1, U.S. Pat. Nos. 6,194,551, and 5,624,821 and 5,648,260. One particular set of substitutions, the triple mutation L234F/L235E/P331S (“TM”) causes a profound decrease in the binding activity of human IgG1 molecules to human C1q, CD64, CD32A and CD16. See, e.g., Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 64:700-704 (2008). In other cases it can be that constant region modifications increase serum half-life. The serum half-life of proteins comprising Fc regions can be increased by increasing the binding affinity of the Fc region for FcRn.

When the antigen-binding protein is an antibody or an antigen-binding fragment thereof, it can further comprise a heavy chain immunoglobulin constant domain selected from the group consisting of: (a) an IgA constant domain; (b) an IgD constant domain; (c) an IgE constant domain; (d) an IgG1 constant domain; (e) an IgG2 constant domain; (f) an IgG3 constant domain; (g) an IgG4 constant domain; and (h) an IgM constant domain. In some embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment thereof that comprises an IgG1 heavy chain immunoglobulin constant domain. In some embodiments, the antigen-binding protein is an antibody or an antigen-binding fragment thereof that comprises an IgG1/IgG3 chimeric heavy chain immunoglobulin constant domain.

The antigen-binding protein of the disclosure (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) can further comprise a light chain immunoglobulin constant domain selected from the group consisting of: (a) an Ig kappa constant domain; and (b) an Ig lambda constant domain.

The antigen-binding protein of the disclosure (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) can further comprise a human IgG1 constant domain and a human lambda constant domain. The antigen-binding protein of the disclosure (including, e.g., anti-O2 antigen antibodies or antigen-binding fragments thereof) can further comprise a human IgG2 constant domain and a human lambda constant domain.

The antigen-binding protein of the disclosure can comprise an IgG1 Fc domain containing a mutation at positions 252, 254 and 256, wherein the position numbering is according to the EU index as in Kabat. For example, the IgG1 Fc domain can contain a mutation of M252Y, S254T, and T256E, wherein the position numbering is according to the EU index as in Kabat.

The present disclosure also relates to an isolated VH domain of the antigen-binding protein of the disclosure and/or an isolated VL domain of the antigen-binding protein of the disclosure.

Antigen-binding proteins (including antibodies or antigen binding fragments thereof) of the disclosure can be labeled with a detectable or functional label. Detectable labels include radiolabels such as 1311 or 99Tc, which may be attached to antibodies of the present disclosure using conventional chemistry known in the art of antibody imaging. Labels also include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g., labeled avidin. Non-limiting examples of other detectable or functional labels which may be attached to the antigen-binding proteins (including antibodies or antigen binding fragments thereof) of the disclosure include: isotopic labels, magnetic labels, redox active moieties, optical dyes, biotinylated groups, fluorescent moieties such as biotin signaling peptides, Green Fluorescent Proteins (GFPs), blue fluorescent proteins (BFPs), cyan fluorescent proteins (CFPs), and yellow fluorescent proteins (YFPs), and polypeptide epitopes recognized by a secondary reporter such as histidine peptide (his), hemagglutinin (HA), gold binding peptide, Flag; a radioisotope, radionuclide, a toxin, a therapeutic and a chemotherapeutic agent.

III. Pharmaceutical Compositions

The disclosure also provides a pharmaceutical composition comprising one or more of the O2-binding agents (including, e.g., anti-O2 antigen antibodies or antigen binding fragments) described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle or pharmaceutically acceptable excipient. In certain embodiments, these pharmaceutical compositions find use in treating, preventing or ameliorating a condition associated with a Klebsiella (e.g., K. pneumoniae) infection in human patients. In certain embodiments, these pharmaceutical compositions find use in inhibiting growth of Klebsiella (e.g., K. pneumoniae). In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O2 serotype. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O1 serotype. In some embodiments, the pharmaceutical composition comprising an O2-binding agent include an anti-O2 antigen antibody or antigen binding fragments thereof that comprise the heavy and light chain complementarity determining region (CDR) sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS30, or KPD1. The CDR sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 antibody as described in Tables 1 and 2 or comprise the variable light chain and variable heavy chain sequences of an anti-O2 antigen KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 antibody as described in Tables 3 and 4.

In certain embodiments, formulations are prepared for storage and use by combining an antibody or anti-O2 binding agent described herein with a pharmaceutically acceptable vehicle (e.g., carrier, excipient) (see, e.g., Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000, herein incorporated by reference). In some embodiments, the formulation comprises a preservative.

The pharmaceutical compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment.

In some embodiments, a pharmaceutical composition comprising one or more of the O2-binding agents (e.g., anti-O2 antigen antibodies or antigen binding fragments) described herein is used for treating pneumonia, urinary tract infection, septicemia/sepsis, neonatal septicemia/sepsis, diarrhea, soft tissue infection, infection following an organ transplant, surgery infection, wound infection, lung infection, pyogenic liver abscesses (PLA), endophthalmitis, meningitis, necrotizing meningitis, ankylosing spondylitis, or spondyloarthropathies. In some embodiments, a pharmaceutical composition comprising one or more of the O2-binding agents (e.g., anti-O2 antigen antibodies or antigen binding fragments) described herein is useful in nosocomial infections, opportunistic infections, infections following organ transplants, and other conditions associated with a Klebsiella infection (e.g. infection with K. pneumoniae, K. oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis, and/or K. granulomatis). In some embodiments, a pharmaceutical composition comprising one or more of the O2-binding agents (including, e.g., anti-O2 antigen antibodies or antigen binding fragments) described herein is useful in subjects exposed to a Klebsiella contaminated device, including, e.g., a ventilator, a catheter, or an intravenous catheter.

In some embodiments, the pharmaceutical composition comprises an amount of an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) that is effective to inhibit growth of the Klebsiella in a subject. In some embodiments, the Klebsiella is K. pneumoniae, K. oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae, K. oxytoca, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O2 serotype. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O1 serotype.

In certain embodiments, the method of treating, preventing, and/or ameliorating Klebsiella infections comprises administering to a subject a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof). In certain embodiments, the subject is a human. In some embodiments, the pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered before the subject is infected with Klebsiella. In some embodiments, the pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered after the subject is infected with a Klebsiella.

In certain embodiments, the pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered to a subject on a ventilator. In certain embodiments, the subject has a catheter (e.g., a urinary catheter or an intravenous catheter). In certain embodiments, the subject is receiving antibiotics (e.g., meropenem, carbapenems, fluoroquinolone, tetracycline, chloramphenicol, trimethoprim, sulfonamide, and/or colistin).

In certain embodiments, a pharmaceutical composition comprising an O2-binding 9agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a Klebsiella infection, wherein the Klebsiella is an extended spectrum beta-lactamase (ESBL) producing Klebsiella. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a Klebsiella infection, wherein the Klebsiella is a non-extended spectrum beta-lactamase (ESBL) producing Klebsiella. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a Klebsiella infection, wherein the Klebsiella is a Klebsiella pneumoniae carbapenemase (KPC) producing Klebsiella.

In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a cephalosporin resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antibody or antigen-binding fragment thereof) is for the treatment or prevention of an aminoglycoside resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a quinolone resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a carbapenem resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a colistin resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of a cephalosporin, aminoglycoside, quinolone, fluoroquinolone, tetracycline, chloramphenicol, trimethoprim, sulfonamide, carbapenem, and colistin resistant Klebsiella infection. In certain embodiments, a pharmaceutical composition comprising an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is for the treatment or prevention of an infection with a Klebsiella that is susceptible to antibiotics.

For the treatment, prevention and/or amelioration of a condition associated with a Klebsiella infection, the appropriate dosage of a pharmaceutical composition, antibody, or anti-O2 binding agent described herein depends on the type of condition, the severity and course of the condition, the responsiveness of the condition, whether the pharmaceutical composition, antibody, or anti-O2 binding agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The pharmaceutical composition, antibody, or anti-O2 binding agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the condition is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual antibody or agent. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. The O2-binding agents and/or pharmaceutical compositions useful to treat, prevent, and/or amelioration of a condition associated with a Klebsiella infection include an anti-O2 antigen antibody or antigen binding fragments thereof that comprise the heavy and light chain complementarity determining region (CDR) sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS30, or KPD1. The CDR sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 antibody as described in Tables 1 and 2 or comprise the variable light chain and variable heavy chain sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 antibody as described in Tables 3 and 4.

IV. Methods of Use

The O2-binding agents (including anti-O2 antigen antibodies and antigen-binding fragments thereof) described herein are useful in a variety of applications including, but not limited to, pneumonia, urinary tract infection, septicemia/sepsis, neonatal septicemia/sepsis, diarrhea, soft tissue infection, infection following an organ transplant, surgery infection, wound infection, lung infection, pyogenic liver abscesses (PLA), endophthalmitis, meningitis, necrotizing meningitis, ankylosing spondylitis, and spondyloarthropathies. In some embodiments, the O2-binding agents (including anti-O2 antigen antibodies and antigen-binding fragments thereof) described herein are useful in nosocomial infections, opportunistic infections, infections following organ transplants, and other conditions associated with a Klebsiella infection (e.g. infection with K. pneumoniae, K oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis, and/or K. granulomatis). In some embodiments, the O2-binding agents (including anti-O2 antigen antibodies and antigen-binding fragments thereof) described herein are useful in subjects exposed to a Klebsiella contaminated device, including, e.g., a ventilator, a catheter, or an intravenous catheter.

In some embodiments, the disclosure provides methods of treating, preventing and/or ameliorating a condition associated with a Klebsiella infection comprising administering an effective amount of an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) to a subject. In some embodiments, the amount is effective to inhibit growth of the Klebsiella in the subject. In some embodiments, the Klebsiella is K. pneumoniae, K oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae, K oxytoca, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O2 serotype. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O1 serotype. In some embodiments, the subject has been exposed to Klebsiella. In some embodiments, Klebsiella has been detected in the subject. In some embodiments, the subject is suspected of being infected with Klebsiella, e.g., based on symptoms.

In some embodiments, the disclosure further provides methods of inhibiting growth of Klebsiella comprising administering an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) to a subject. In some embodiments, the Klebsiella is K. pneumoniae, K. oxytoca, K. planticola, K. ozaenae, K. rhinosclermoatis, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae, K. oxytoca, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O2 serotype. In some embodiments, the Klebsiella (e.g., K. pneumoniae) is of the O1 serotype. In some embodiments, the subject has been exposed to Klebsiella. In some embodiments, Klebsiella has been detected in the subject. In some embodiments, the subject is suspected of being infected with a Klebsiella, e.g., based on symptoms.

In some embodiments, the methods of treating, preventing and/or ameliorating a condition associated with a Klebsiella infection comprises contacting a subject infected with a Klebsiella with the O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) in vivo. In certain embodiments, contacting a cell with an O2-binding agent is undertaken in an animal model. For example, O2-binding agents can be administered to murine Klebsiella infection models to reduce bacterial burden. In some embodiments, the O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered before introduction of bacteria to the animal to prevent infections. In some embodiments, the O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered at the same time or shortly after the animal has been exposed to bacteria to prevent infection. In some embodiments, the O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) is administered as a therapeutic after infection.

In certain embodiments, the subject is on a ventilator. In certain embodiments, the subject has a catheter (e.g., a urinary catheter or an intravenous catheter). In certain embodiments, the subject is receiving antibiotics (e.g., meropenem, carbapenems, or colistin).

In certain embodiments, the Klebsiella infection is a nosocomial infection. In certain embodiments, the Klebsiella infection is an opportunistic infection. In certain embodiments, the Klebsiella infection follows an organ transplant.

In certain embodiments, the Klebsiella is an extended spectrum beta-lactamase (ESBL) producing Klebsiella. In certain embodiments, the Klebsiella is a non-ESBL producing Klebsiella. In certain embodiments, the Klebsiella is a Klebsiella pneumoniae carbapenemase (KPC) producing Klebsiella.

In certain embodiments, the Klebsiella is cephalosporin resistant. In certain embodiments, the Klebsiella is aminoglycoside resistant. In certain embodiments, the Klebsiella is quinolone resistant. In certain embodiments, the Klebsiella is carbapenem resistant. In certain embodiments, the Klebsiella is cephalosporin, aminoglycoside, quinolone, and carbapenem resistant. In certain embodiments, the Klebsiella is cephalosporin, aminoglycoside, and quinolone resistant. In certain embodiments, the Klebsiella is susceptible to antibiotics.

In certain embodiments, the method of treating, preventing, and/or ameliorating Klebsiella infections comprises administering to a subject an effective amount of an O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) and an antibiotic. The O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) and the antibiotic can be administered simultaneously or sequentially. The O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) and the antibiotic can be administered in the same pharmaceutical composition. The O2-binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) and the antibiotic can be administered in separate pharmaceutical compositions simultaneously or sequentially. In certain embodiments, the antibiotic is an antibiotic suitable to treat a Klebsiella infection. In certain embodiments, the antibiotic is meropenem. In certain embodiments, the antibiotic is a carbapanem or colistin. In certain embodiments, the antibiotic is a cephalosporin, aminoglycoside, quinolone, fluoroquinolone, tetracycline, chloramphenicol, trimethoprim, sulfonamide, carbapenem, and/or colistin.

The present disclosure also provides methods of detecting O2 lipopolysaccharide or Klebsiella containing O2 antigen. In some embodiments, a method of detecting O2 or Klebsiella containing O2 antigen comprises contacting a sample with an O2 binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) provided herein and assaying for binding of the binding agent (e.g., an antibody or antigen-binding fragment thereof) to the sample. Methods of assessing binding are well known in the art. In some embodiments, the methods comprise detecting O1 lipopolysaccharide or Klebsiella containing O1 antigen and O2 lipopolysaccharide or Klebsiella containing O2 antigen. In some embodiments, a method of detecting O1 and O2 or Klebsiella containing O1 or O2 antigen comprises contacting a sample with an O2 binding agent (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) provided herein and assaying for binding of the binding agent (e.g., an antibody or antigen-binding fragment thereof) to the sample. Methods of assessing binding are well known in the art.

V. Kits

A kit comprising an isolated antigen-binding protein (e.g. an anti-O2 antigen antibody or antigen-binding fragment thereof) according to any aspect or embodiment of the present disclosure is also provided as an aspect of the present disclosure. In a kit, the antigen-binding protein, antibody, or antigen-binding fragment thereof can be labeled to allow its reactivity in a sample to be determined, e.g., as described further below. Components of a kit are generally sterile and in sealed vials or other containers. Kits can be employed in diagnostic analysis or other methods for which antibodies are useful. A kit can contain instructions for use of the components in a method, e.g., a method in accordance with the present disclosure. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the disclosure. O2-binding agents suitable for use in a kit include an anti-O2 antigen antibodies or antigen binding fragments thereof that comprise the heavy and light chain complementarity determining region (CDR) sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1. The CDR sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS30, and KPD1 antibodies as described in Tables 1 and 2 or comprise the variable light chain and variable heavy chain sequences of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, and KPD1 antibodies as described in Tables 3 and 4.

The reactivities of antibodies or antigen-binding fragments thereof in a sample can be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility. Radioactive labeled antigen is mixed with unlabeled antigen (the test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay can also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule can be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules can be enzymes which catalyze reactions that develop or change colors or cause changes in electrical properties, for example. They can be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They can include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems can be employed.

The signals generated by individual antibody-reporter conjugates can be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test).

The present disclosure also provides the use of an antigen-binding protein as described above for measuring antigen levels in a competition assay, including methods of measuring the level of O2 antigen or Klebsiella containing O2 antigen in a sample by employing an antigen-binding protein provided by the present disclosure in a competition assay. In some embodiments, the physical separation of bound from unbound antigen is not required. In some embodiments, a reporter molecule is linked to the antigen-binding protein so that a physical or optical change occurs on binding. The reporter molecule can directly or indirectly generate detectable, and preferably measurable, signals. In some embodiments, the linkage of reporter molecules is direct or indirect, or covalent, e.g., via a peptide bond or non-covalent interaction. Linkage via a peptide bond can be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

The present disclosure also provides methods of measuring levels of O2 antigen directly, by employing an antigen-binding protein (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) according to the disclosure. In some embodiments, these methods utilize a biosensor system. In some embodiments, the methods comprise detecting O1 and O2 antigen by employing an antigen-binding protein (e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof) according to the disclosure. In some embodiments, these methods utilize a biosensor system.

VI. Polynucleotides and Host Cells

In further aspects, the present disclosure provides an isolated nucleic acid comprising a nucleic acid sequence encoding an antigen-binding protein (e.g. an anti-O2 antigen antibody or antigen-binding fragment thereof), VH domain and/or VL domain according to the present disclosure. In some aspects the present disclosure provides methods of making or preparing an antigen-binding protein (e.g. an anti-O2 antigen antibody or antigen-binding fragment thereof), a VH domain and/or a VL domain described herein, comprising expressing said nucleic acid under conditions to bring about production of said antigen-binding protein, VH domain and/or VL domain and, optionally, recovering the antigen-binding protein, VH domain and/or VL domain.

A nucleic acid provided by the present disclosure includes DNA and/or RNA. In one aspect, the nucleic acid is cDNA. In one aspect, the present disclosure provides a nucleic acid which codes for a CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site or antibody, e.g., scFv, IgG1, or IgG2, as described above (see, e.g., Tables 1-4).

One aspect of the present disclosure provides a nucleic acid, generally isolated, optionally a cDNA, encoding a VH CDR or VL CDR sequence described herein. In some embodiments, the VH CDR sequence is selected from the SEQ ID NOs provided in Table 1. In some embodiments, the VL CDR sequence is selected from the SEQ ID NOs provided in Table 2. A nucleic acid encoding the KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-538Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 set of HCDRs and nucleic acid encoding the KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 set of LCDRs are also provided, as are nucleic acids encoding individual CDRs, HCDRs, LCDRs and sets of CDRs, HCDRs, LCDRs as described in Tables 1 and 2. In some embodiments, the nucleic acids of the present disclosure encode a VH and/or VL domain of KPN42, KPN42-v2016, KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL, KPS3, KPN70, KPN179, KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL, KPN179-FR-GL N35S-VH/KPN179-FR-GL-C105A-VL, KPN44, KPN17, 6F6, KPL26, KPS18, KPS24, KPS44, KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL (KPS44-v2017), KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3), KPS30, or KPD1 as described in Tables 3 and 4.

The invention further provides a polynucleotide comprising a sequence selected from those shown in Tables 5 and 6 below.

TABLE 5

Variable heavy chain polynucleotide sequences

Antibody
VH Polynucleotide Sequence (SEQ ID NO)

KPN42
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCGGGGGGG

TCCCTTAGACTCTCCTGTGCAGCCTCTGGTTTCACTTTCAATGACGCCTGG

ATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGGCTGGAGTGGGTCGCCC

GCATTAAAAAGAAACATGAAGGTGTTACGACAGACTACCCTGCATCCGT

GAGAGGCAGATTCACCATCTCAAGAGATGATTCTAAAAACACAGTGTAT

CTGCAGATGGGCAGACTGAGAATCGAGGACACTGCCATATATTACTGTA

CCACAAGGATAGTGACTACCAATGACTACTGGGGCCAGGGAACCCTGGT

CACCGTCTCCTCAG (SEQ ID NO: 136)

KPN42-v2016
GAGGTGCAGCTGGTCGAATCTGGCGGGGGACTGGTGAAACCTGGCGGCT

CTCTGAGGCTGAGTTGCGCCGCTTCAGGCTTCACCTTCAACGACGCATGG

ATGAATTGGGTGCGACAGGCACCTGGAAAGGGACTGGAGTGGGTCGGCC

GGATCAAGAAAAAGCACGAAGGGGTGACCACAGATTACCCTGCTAGCGT

CCGGGGAAGATTCACTATTAGCAGAGACGATTCCAAAAACACCGTGTAT

CTGCAGATGGGCAGGCTGCGCATCGAGGACACCGCCATCTACTATTGTA

CTACCCGCATCGTGACAACTAATGATTACTGGGGGCAGGGAACCCTGGT

GACAGTCAGCTCC (SEQ ID NO: 137)

KPN42-FR-GL-
GAGGTGCAGCTGGTCGAATCTGGCGGGGGACTGGTGAAGCCTGGCGGCT

VH/KPN42-FR-
CTCTGCGACTGAGTTGCGCCGCTTCAGGCTTCACCTTTAACGACGCTTGG

GL-C105A-VL
ATGAATTGGGTGAGGCAGGCACCTGGAAAAGGACTGGAGTGGGTGGGA

CGCATCAAGAAAAAGCACGAAGGGGTGACCACAGATTACGCAGCCCCT

GTCAAAGGCCGGTTCACAATTAGCAGAGACGATTCCAAGAACACTCTGT

ATCTGCAGATGAATAGCCTGAAAACCGAGGACACAGCCGTGTACTATTG

TACTACCAGAATCGTCACAACTAACGATTACTGGGGGCAGGGAACTCTG

GTGACCGTCAGCTCC (SEQ ID NO: 138)

KPS3
CAGGGACAGTTGGTGGACTCTGGGGGAGGCGTGGTCCAGCGGGGGGGG

TCTCAGAGACTCTCCTGCGCAGCGTCTGGATTCAGCTTCAGAGACTATGG

CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCC

TTTATATCATATGATGGGAGAGATCAATACTATGCAGACTCCGTGAAGG

GCCGATTCATCATCTCCAGAGACAATTCCAAGAACACGCTGTCTCTGCAA

ATGAACAGCCTGAGACCTGAGGACACGGCTGTCTATTACTGTGGGCCTTT

TTATAACCCCAGTCTCTACTACCCCCCCTGGGGCCACGGACTTCCGGTCA

TCGTCTCCTCAG (SEQ ID NO: 139)

KPN70
CAGGTGCAGCTGCAGGAGTCGGGCCCGGGACTGGTGAAGCCTTCGGAGA

CCCTGTCTCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTACTTACTACT

GGAACTGGATCCGGCAGTCCCCAGGGAAGGAATTGGAGTGGATTGCAAA

TATACATCAAAGTGGGACCACCTACTACAACCCCTCCCTCAAGAGTCGA

GTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGGTGA

TCTCTGTGACTGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGAGTCC

GACGATGGCTACAAGTGGAACTACTTTGACTACTGGGGCCAGGGAACCC

TAGTCACCGTCTCCTCAG (SEQ ID NO: 140)

KPN179
GAGGTGCAGGTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCGGGGGGG

TCCCTTAGACTCTCCTGTGCAGCCTCTGGTTTCACTTTCAATAACGCCTGG

ATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCGGCC

GTATTAAAAGGAAAGCTGATGGTGAGACAACAGACTACCCTGCATCCGT

GAAAGGCAGATTCACCGTCTCAAGAGATGATTCAAAAAACACGATATAT

CTGCAGATGAACAGCCTGAAAACCGAGGACACAGCCATATATTACTGTA

CCACAAGGATAGTGACTACCAATGACTACTGGGGCCAGGGAACCCTGGT

CACCGTCTCCTCAG (SEQ ID NO: 141)

KPN179-FR-1-2-
GAGGTGCAGCTGGTCGAATCCGGCGGGGGACTGGTGAAACCTGGCGGCT

4-GL-N35S-
CTCTGCGACTGAGTTGCGCCGCTTCAGGCTTCACCTTTAGCAACGCATGG

VH/KPN179-FR-
ATGAATTGGGTGAGACAGGCACCTGGAAAGGGACTGGAGTGGGTCGGC

GL-C105A-VL
CGGATCAAGAGAAAAGCTGACGGGGAAACCACAGATTACCCTGCATCTG

TGAAGGGCAGGTTCACAGTCAGCCGCGACGATTCCAAAAACACTATCTA

CCTGCAGATGAATAGTCTGAAGACCGAGGACACAGCCATCTACTATTGT

ACTACCCGGATTGTGACAACTAACGATTACTGGGGGCAGGGAACTCTGG

TGACCGTCAGCTCC (SEQ ID NO: 193)

KPN179-FR-GL
GAGGTGCAGCTGGTCGAATCTGGCGGGGGACTGGTGAAACCTGGCGGCT

N35S-
CTCTGCGACTGAGTTGCGCCGCTTCAGGCTTCACCTTTAGCAACGCTTGG

VH/KPN179-FR-
ATGAATTGGGTGAGACAGGCACCTGGAAAGGGACTGGAGTGGGTGGGA

GL-C105A-VL
CGGATCAAGAGAAAAGCCGACGGGGAAACCACAGATTACGCAGCCCCT

GTGAAGGGCAGGTTCACAATTAGCCGCGACGATTCCAAAAACACTCTGT

ATCTGCAGATGAATAGCCTGAAGACCGAGGACACAGCCGTGTACTATTG

TACTACCCGGATCGTCACAACTAACGATTACTGGGGGCAGGGAACTCTG

GTGACCGTCAGCTCC (SEQ ID NO: 194)

KPN44
CAGGTGCAGCTGCAGGAGTCGGGCCCGGGACTGGTGAAGCCTTCGGAGA

CCCTGTCTCTCACCTGCACTGTGTCCGGTGGCTCCACCAGTAGTTACTAC

TGGAACTGGATCCGGCAGGCCCCAGGGAAGCCATTGCAGTGGATTGCAA

ATATACATCACGGTGGGACCACTTATTACAACCCCTCCCTCAGGAGTCGG

GTCACCATGTCTCTGGACACTTCCAATAACCAGTTCTCCCTGAAGCTGAC

CTCTGTGACTGCTGCGGACACGGCCGTCTATTTCTGTGCGAGAGAGTCCG

ACGATGGCTACAGGTGGAACTACTTTGACTACTGGGGCCAGGGAGTCCT

GGTCACCGTCTCCTCAG (SEQ ID NO: 142)

KPN17
GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCTTGGTTCAGCCTGGGGGGT

CCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCACTTCTGG

ATGCACTGGGTCCGCCAAGCTCCAGGGCAGGGGCTGGTGTGGGTCGCAC

GTATTGATGGTTCTGTGACAAACTTGAGGTACGCGGGCTCCGTGGAGGG

GCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATTTGCAA

ATGAACAGTCTGAGAGACGAGGACACGGCTGTATATTACTGTGCAAGAG

ATTTGGTAGGAATTGGCACGCCGGCCGGGTACGGTATGGACGTCTGGGG

CCAAGGGACCACGGTCACCGTCTCCTCAG (SEQ ID NO: 143)

6F6
CAGGTTCACCTACAACAGTCTGGTTCTGAACTGAGGAGTCCTGGGTCTTC

AGTAAAGCTTTCATGCAAGGATTTTGATTCAGACGTCTTCCCTATTGCTT

ATATGGGTTGGATTAGGCAGCAGCCTGGGCATGGATTTGACTGGATTGG

GGACATACTCCCAAATATTGGTAGAACAATCTATGGAGAGAAGTTTGAG

GACAAAGCCACACTGGATGCAGACACAGTGTCCAACACAGCCTACTTGG

AGCTCAGCAGTCTGACATCTGAGGACTCTGCTATCTACTATTGTGCAAGG

AGGGGGACGTCGGGGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCA

CCGTCTCCTCA (SEQ ID NO: 144)

KPL26
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGTCGGGGGGGT

CCCTGAGACTCTCCTGTGAAACCTCTGGATTCATTTTTGGTAGTTCTTGG

ATGACCTGGGTCCGCCAGGCTCCAGGGAAAGGGCTGGAGTGGGTGGCCA

CCATAAACCCTGATGGAAGTGCGACAAGCTATGAGGACTCTGTGAGGGG

CCGATTCGCCGTCTCCAGAGACAACGCCAAGAACTCAGTGTATCTGCAA

ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTACTTCTGTACAAGGA

ATAAGGCATACAATGCCCTTGACTACTGGGGCCAGGGAACCCTGGTCAC

CGTCTCCTCAG (SEQ ID NO: 145)

KPS18
GAGGTTCGCCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGT

CCCTAAGACTCTCCTGTGCAGCCTCAGGATTCACTTTCAAAAACGCCTGG

ATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCC

GTGTTAAAAACGAAGTTGATGGGGGGACAATAGACTACGGTGTGCCCGT

GAGAGGCAGATTCACCATCTCAAGAGACGATTCACAAGGCACGCTGTCT

CTGGAGATGAACAGCCTGAGAGAGGATGACACAGGGATTTATTACTGTC

GGGCTTTTTGGAGTGGTTTTCCTGCCGGATACTGGGGCCAGGGAACCCTG

GTCAGCGTCTCCTCAG (SEQ ID NO: 146)

KPS24
GAGCTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGT

CCCTTAGACTCTCCTGTGCAGCCTCAGGATTCACTTTCAAAAACGCCTGG

ATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCC

GTGTTAAAAGCGAAGTTGATGGGGGGACAACAGACTACGGTGTGCCCGT

GAGAGGCAGATTCACCATCTCAAGAGATGATTCACAAAGCACGCTGTCT

CTGGAGATGAGCAGCCTGCAAGACGATGACACAGGCGTTTATTACTGTC

GGGCTTTTTGGAGTGATTTTCAAACCGGCTACTGGGGCCAGGGAACCCT

GGTCACCGTCTCCTCAG (SEQ ID NO: 147)

KPS44
GAGGTGCACCTGGTGGAGTCTGGGGGAGGCTTGGTAAAGCCTGGGGGGT

CCCTTAGACTCTCCTGTGCAGCCTCAGGATTCACTTTCAAAAACGCCTGG

ATGAGCTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCC

GTGTTAAAAGCGAAGTTGATGGGGGGACAATAGACTACGGTGTGCCCGT

GAGAGGCAGATTCACCATCTCAAGAGATGATTCACAAGGCACACTGTCT

CTGGAGATGAACAGCCTGAAAGACGATGACACAGGCGTTTATTATTGTC

GGGCTTTTTGGAGTGGTTTTCCTACCGGATACTGGGGCCAGGGAGCCCTG

GTCAGCGTCTCCTCAG (SEQ ID NO: 148)

KPS44-v2017
GAGGTGCACCTGGTCGAATCCGGCGGGGGACTGGTGAAACCAGGCGGGT

CTCTGAGACTGAGTTGCGCCGCTTCAGGCTTCACCTTCAAGAACGCATGG

ATGAGCTGGATTAGACAGGCACCTGGGAAGGGACTGGAGTGGGTGGGC

CGCGTCAAATCTGAAGTGGATGGAGGCACCATCGACTACGGGGTGCCTG

TCCGGGGAAGATTCACCATTAGCCGAGACGATTCCCAGGGCACACTGTC

TCTGGAGATGAATAGTCTGAAGGACGATGACACTGGGGTGTACTATTGT

AGAGCTTTCTTTTCAGGATTTCCTACCGGCTATTGGGGACAGGGGGCCCT

GGTGAGCGTCAGCTCC (SEQ ID NO: 206)

KPS44-G1
GAGGTACACCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGGGGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTCAGGGTACACTTAG

CCTCGAAATGAATAGCCTCAAAGACGATGATACAGGCGTTTATTATTGC

CGCGCATTCTGGAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTGCTC

TTGTCTCAGTGTCATCC (SEQ ID NO: 207)

KPS44-G2
GAGGTACACCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGGGGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTCAGGGTACACTTAG

CCTCGAAATGAATAGCCTCAAAGACGATGATACAGGCGTTTATTATTGC

CGCGCATTCTTTAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTGCTCT

TGTCTCAGTGTCATCC (SEQ ID NO: 208)

KPS44-G3
GAGGTACACCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGGGGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTCAGGGTACACTTAG

CCTCGAAATGAATAGCCTCAAAGACGATGATACAGGCGTTTATTATTGC

CGCGCATTCTTTAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTGCTCT

TGTCTCAGTGTCATCC (SEQ ID NO: 208)

KPS30
GAGATGCAGTTGGTAGAGTCTGGGGGAGGCTTGGTCCAGCCTGGGGTGT

CCCTGAGACTCTCCTGTGTAGACTCTGGATTCAGTTTTAGTACCTCTTGGT

TGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGCTGGCCAA

CATAGATCCAGATGGAAGCACGAGAAATCATGTGGACTCTGTGAGGGGC

CGATTCACCATCTCCAGAGACAACGCCAAGAATTCACTGTATCTCCAGAT

GAACAGCCTGAGAGCCGAGGACACGGCCGTCTATTACTGTGCGAGAGAC

TATGCCTACAATCGCTTTGACTACTGGGGCCAGGGAACCATGGTCACCGT

CTCCTCAG (SEQ ID NO: 149)

KPD1
CAGGTGCAGCTGCAGGAGTCGGACCCACGACTGGTGAAGCCTTCGGAGA

CCCTGTCCCTCACCTGTAGTGTCTCTGGTGTCTCCATCACCAGTAACACTT

ACTGGTGGGCCTGGATCCGCCAGCCCCCAGGGAAGAAACTGGAGTGGAT

TGGGAGTCTCTCTTACAGTGGGGACACCTACTACAACCCGTCCCTCACGA

GTCGCGTCACCATATCAAGAGATATCCATCAGAACCAATTTTTCCTGGAG

TTGAACTCTGTGACCGCCGCCGACACGGCCATGTATTACTGTGCGAGAG

ATCCCGACATCATTCGCAATTTCCAGTTTGACTACTGGGGCCGGGGAACC

CTGGTCACCGTCTCCTCGG (SEQ ID NO: 150)

KPL36
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGTCTGGGGGGT

CCCTGAGACTCTCCTGTGAGACTTCTGGATTCACCTTTATAAGTTCTTGG

ATGAGTTGGGTCCGCCAGGCTCCAGGGACAGGACTGGAGTGGGTGGCCA

CCATTAACCCTGATGGAACTGAGACACCCTACGCGGACTCGCTGAAGGG

CCGCTTCACCATCTCCAGAGACAACACCAAGAAGTCACTTTATCTGCAA

ATCCATAGCCTGAGAGCCGACGACACGGCCGTCTATTTCTGTGCAAGGA

ATAAGGCATACAATGCCCATGACTTCTGGGGCCAGGGAACCCTGGTCAC

CGTCTCCTCAG (SEQ ID NO: 195)

KPS44-G4
CAGGTACAGCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCCGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTAAGAATACACTTTA

CCTCCAGATGAATAGCCTCAAAACCGAGGATACAGCCGTTTATTATTGCC

GCGCATTCTATAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTACTCTT

GTCACAGTGTCATCC (SEQ ID NO: 221)

KPS44-G6
CAGGTACAGCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCCGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTAAGAATACACTTTA

CCTCCAGATGAATAGCCTCAAAACCGAGGATACAGCCGTTTATTATTGCC

GCGCATTCTATAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTACTCTT

GTCACAGTGTCATCC (SEQ ID NO: 231)

KPS44-G8
CAGGTACAGCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCCGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTAAGAATACACTTTA

CCTCCAGATGAATAGCCTCAAAACCGAGGATACAGCCGTTTATTATTGCC

GCGCATTCTATAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTACTCTT

GTCACAGTGTCATCC (SEQ ID NO: 241)

KPS44-G10
GAGGTACACCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCGGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTCAGGGTACACTTAG

CCTCGAAATGAATAGCCTCAAAGACGATGATACAGGCGTTTATTATTGC

CGCGCATTCTACAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTGCTCT

TGTCTCAGTGTCATCC (SEQ ID NO: 251)

KPS44-G11
GAGGTACACCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCGGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTCAGGGTACACTTAG

CCTCGAAATGAATAGCCTCAAAGACGATGATACAGGCGTTTATTATTGC

CGCGCATTCTACAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTGCTCT

TGTCTCAGTGTCATCC (SEQ ID NO: 261)

KPS44-G14
CAGGTACAGCTTGTAGAAAGTGGGGGTGGGCTTGTCAAGCCTGGGGGAA

GTTTGAGACTGAGTTGCGCCGCAAGTGGCTTCACGTTTAAGAACGCATG

GATGTCCTGGATTAGACAAGCCCCCGGTAAAGGTTTGGAATGGGTAGGA

CGAGTTAAGTCTGAGGTTGACGCCGGGACGATAGATTACGGTGTTCCCG

TGCGCGGCAGATTCACGATAAGTCGAGACGACTCTAAGAATACACTTTA

CCTCCAGATGAATAGCCTCAAAACCGAGGATACAGCCGTTTATTATTGCC

GCGCATTCTATAGTGGCTTCCCGACTGGGTACTGGGGGCAAGGTACTCTT

GTCACAGTGTCATCC (SEQ ID NO: 271)

TABLE 6

Variable light chain polynucleotide sequences

Antibody
VL Polynucleotide Sequence (SEQ ID NO)

KPN42
CAGTCTGCCCTGACTCAGCCTCCCTCAGTGTCCGGGTCTCCTGGACAGTC

AGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGCTTACGACT

ATGTCTCCTGGTACCAACAGTACGCAGGCAAAGTCCCCAAACACATAAT

TTATGATGTCAATGAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCT

CCAAGTCTGGCAACACGGCCGCCCTGACCATCTCTGGGCTCCAGGCTGA

GGATGAGGCTGATTATTATTGCTGCTCATATGCAGGCGGTGACATCTTTG

TCTTCGGAACTGGGACTCAGGTCACCGTCCTA (SEQ ID NO: 151)

KPN42-v2016
CAGTCTGCCCTGACCCAGCCTAGGTCTGTGAGTGGGTCACCCGGACAGA

GTGTCACAATCTCATGCACCGGAACAAGCTCCGACGTGGGCGCTTACGA

TTATGTCTCTTGGTACCAGCAGCACCCCGGGAAGGCACCTAAACTGATG

ATCTACGACGTGAACAAGCGGCCAAGTGGCGTCCCCGATAGATTCAGCG

GCTCCAAATCTGGGAATACAGCTAGCCTGACTATCTCCGGCCTGCAGGC

AGAGGACGAAGCCGATTACTATTGTGCCAGCTACGCTGGCGGGGACATT

TTCGTGTTTGGAACTGGCACCAAGGTGACCGTCCTG (SEQ ID NO: 152)

KPN42-FR-GL-
CAGTCTGCCCTGACCCAGCCTAGGTCTGTGAGTGGGTCACCCGGACAGA

VH/KPN42-FR-
GTGTCACAATCTCATGCACCGGAACAAGCTCCGACGTGGGCGCTTACGA

GL-C105A-VL
TTATGTCTCTTGGTACCAGCAGCACCCCGGGAAGGCACCTAAACTGATG

ATCTACGACGTGAACAAGCGGCCAAGTGGCGTCCCCGATAGATTCAGCG

GCTCCAAATCTGGGAATACAGCTAGCCTGACTATCTCCGGCCTGCAGGC

AGAGGACGAAGCCGATTACTATTGTGCCAGCTACGCTGGCGGGGACATT

TTCGTGTTTGGAACTGGCACCAAGGTGACCGTCCTG (SEQ ID NO: 153)

KPS3
GAGGTTGTCTTGACACAGTCTCCAGCCACTCTGTCTTTGTCTCCAGGGGA

AAGAGCCACCCTCTCCTGTAGGGCCAGTCAGAGCATTAGCAGCCAATTA

GCGTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCCATG

ATGCATCCAACAGGGACACTGGCGTCCCAGACAGGTTCAGTGGCAGTGG

GTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGAT

TTTGCTATGTATTACTGTCTCCAGCGTAACAACTGGCCTCCGTGGACGTT

CGGCCAAGGGACCAAGGTGGAAATCAAAC (SEQ ID NO: 154)

KPN70
GAAATTGTGTTGACACAGTCTCCAGCCTCCCTGTCTTTGTCTCCAGGGGA

AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGATTGTTACCAACTACTTAG

CCTGGTATCAACATAAACCTGGCCAGGCTCCCAGGCTCCTCATCTTTGAT

ATGTCCATTAGGGCCGCTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGT

CTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTTT

GCAGTTTATTACTGTCAACACCGTAGCAACTGGCCTCTATTCACTTTCGG

CCCTGGGACCAAAGTGGATATCAAAC (SEQ ID NO: 155)

KPN179
CAGTCTGCCCTGACTCAGCCTCCCTCAGTGTCCGGGTCTCCTGGACAGTC

AGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTTATTACGACT

ATGTCTCCTGGTACCAACAGCACCACCCAGGCAAAGCCCCCAAACACAT

GATTTATGATGTCAATAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTG

GCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCT

GAGGATGAGGCTGATTATTATTGCTGTTCATATGCAGGCGGTGACACTTT

TGTCTTCGGAACTGGGACCAAGGTCACCGTCCTAG (SEQ ID NO: 156)

KPN179-FR-1-2-
CAGTCTGCCCTGACTCAGCCTAGGTCTGTGAGTGGGTCACCCGGACAGA

4-GL-N35S-
GTGTCACAATCTCATGCACCGGAACAAGCTCCGACGTGGGCTACTATGA

VH/KPN179-FR-
TTACGTCTCTTGGTATCAGCAGCACCCCGGGAAGGCTCCTAAACTGATGA

GL-C105A-VL
TCTACGACGTGAACAAGCGGCCAAGTGGCGTCCCCGATAGATTCAGCGG

CTCCAAATCTGGGAATACAGCAAGCCTGACTATTTCCGGCCTGCAGGCA

GAGGACGAAGCCGATTACTATTGTGCCAGCTATGCTGGCGGGGACACCT

TCGTGTTTGGAACTGGCACCAAGGTGACAGTCCTG (SEQ ID NO: 196)

KPN179-FR-GL
CAGTCTGCCCTGACTCAGCCTAGGTCTGTGAGTGGGTCACCCGGACAGA

N35S-
GTGTCACAATCTCATGCACCGGAACAAGCTCCGACGTGGGCTACTATGA

VH/KPN179-FR-
TTACGTCTCTTGGTATCAGCAGCACCCCGGGAAGGCTCCTAAACTGATGA

GL-C105A-VL
TCTACGACGTGAACAAGCGGCCAAGTGGCGTCCCCGATAGATTCAGCGG

CTCCAAATCTGGGAATACAGCAAGCCTGACTATTTCCGGCCTGCAGGCA

GAGGACGAAGCCGATTACTATTGTGCCAGCTATGCTGGCGGGGACACCT

TCGTGTTTGGAACTGGCACCAAGGTGACAGTCCTG (SEQ ID NO: 197)

KPN44
GAAATTGTGTTGACACAGTCTCCAGCCTCCCTGTCTTTGTCTCCAGGGGA

CAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACGATTACCAACTACTTA

GCCTGGTACCAACATAAACCTGGCCAGGCTCCCAGACTCCTCATCTTTGA

TATGTCGAAAAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGG

TCTGGGACAGACTTCACTCTCACCATCAGCAGCCTTGAGCCTGAAGATTT

TGCAGTTTACTACTGTCAACACCGTAGCAACTGGCCTCTATTCACTTTCG

GCCCTGGGACCAACGTGGATATCAAAC (SEQ ID NO: 157)

KPN17
GACATCCAGTTGACCCAGTCTCCATCCTTCCTGTCTGCCTCTGTAGGAGA

CAGAGTCACCATCACTTGCCGGGCCAGTCAGGGCATTAGCACTTATTTAG

CCTGGTATCAACAAAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGC

TGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGA

TCTGGGACAGAATTCACTCTCACAATCAACAGCCTGCAGTCTGAAGATTT

TGCAACTTACTACTGTCAGCAGCTTACTAGTCACCTCTACACTTTTGGCC

AGGGGACCAAGCTGGAGATCAAAC (SEQ ID NO: 158)

6F6
GATGTTGTGATGACCCAAACTCCACTCTTCCTGCCTGTCAGTCTTGGAGA

TCAAGCCTCCATCTCTTGCAGATCTAGTCAGGGCCTTGTACACAGTACTG

GAAACACCTTTTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAA

GCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGT

TCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGT

GGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATATTC

CGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID

NO: 159)

KPL26
CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAATC

AGTCACCCTCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTAATAACT

ATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATCAT

TTATGAGGTCAGTAAGCGGCCCTCAGGGGTCCCTAATCGTTTCTCTGGCT

CCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGA

GGATGAGGCTGATTATTACTGCAGCTCATTTGGAGGTAGTAAGATGTTCG

GCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 160)

KPS18
CAGTCTGTGTTGACGCAGCCGCCCTCACTGTCTGCGGCCCCAGGACAGA

CGGTCACCATCGCCTGCTCTGGAAGTAGATCCAACATTGGGAGTGATTCC

GTCTCCTGGTTCCAGCAGTTCCCAGGAACAGCCCCCAGAGTCCTCATGTA

TGACAATAATAAGCGACCCTCAGGCATTTCTGACCGATTCTCTGGCTCCA

AGTCTGGCACGTCAGTCACCCTGGACATCACCGGACTCCAGACTGGGGA

CGAGGCCGATTATTACTGCGCAACATGGGATAGCAGCCTGAGTGCTTAT

GTCTTCGGATCTGGGACCAAGGTCACCGTCCTAA (SEQ ID NO: 161)

KPS24
CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGA

CGGTCACCATCGCCTGCTCTGGAAGTAGCTCCAACATTGGGAGTGATTCC

GTATCCTGGTTCCAGCAGCTCCCAGGAACAGCCCCCAGAGTCCTCATGTA

TGAAAATAATAAGCGACCCTCAGGGATTTCTGACCGATTCTCTGGCTCCA

AGTCTGGCACGTCAGTCACCCTGGGCATCACCGGACTCCAGACTGGGGA

CGAGGCCGATTATTACTGCGCAGCATGGGATAGCAGCCTACGTGCTTAT

GTCTTCGGATCTGGGACCAAGGTCACCGTCCTAG (SEQ ID NO: 162)

KPS44
CAGTCTGTGTTGACGCAGCCGCCCTCACTGTCTGCGGCCCCTGGACAGAC

GATCACCATCGCCTGCTCTGGAACTAGTTCCAACATTGGGAGTGATTCCG

TATCCTGGTTCCAGCAATTCCCAGGAACAGCCCCCAGAGTCCTCATATAT

GAGAATAATAAGCGACCCTCAGGCATTTCTGACCGATTCTCTGGCTCCAA

GTCTGGCACGTCAGTCACACTGGGCATCACCGGACTCCAGACTGGGGAC

GAGGCCGATTATTACTGCGCAACATGGGATAGCAGCCTGAGTGCTTATG

TCTTCGGATCTGGGACCAAGGTCACCGTCCTAG (SEQ ID NO: 163)

KPS44-v2017
CAGAGCGTGCTGACACAGCCCCCTTCACTGAGCGCCGCTCCTGGACAGA

CCATCACAATTGCTTGCTCCGGCACTAGCTCCAACATCGGGTCCAATTAC

GTGTCTTGGTTCCAGCAGTTTCCAGGAACCGCACCCAGGGTCCTGATCTA

TGAGAACAATAAGCGGCCCTCAGGCATTAGCGACAGATTCTCCGGGTCT

AAAAGTGGAACTAGCGTGACCCTGGGAATTACCGGCCTGCAGACAGGCG

ACGAAGCAGATTACTATTGTGCCACCTTCGATTCTAGTCTGAGTGCCTAC

GTCTTTGGCTCTGGGACAAAAGTGACTGTCCTG (SEQ ID NO: 209)

KPS44-G1
CAGTCCGTTTTGACGCAACCCCCGTCACTGAGTGCTGCGCCTGGGCAGAC

CATAACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTAATTAT

GTATCATGGTTCCAGCAATTCCCTGGCACGGCACCTCGCGTACTGATCTA

CGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAGC

AAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGTG

ATGAAGCTGATTACTACTGCGCTACTTTTGATAGCTCTCTTTCAGCTTAC

GTGTTTGGTTCCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 210)

KPS44-G2
CAGTCCGTTTTGACGCAACCCCCGTCACTGAGTGCTGCGCCTGGGCAGAC

CATAACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATTCT

GTATCATGGTTCCAGCAATTCCCTGGCACGGCACCTCGCGTACTGATCTA

CGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAGC

AAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGTG

ATGAAGCTGATTACTACTGCGCTACTTTTGATAGCTCTCTTTCAGCTTAC

GTGTTTGGTTCCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 211)

KPS44-G3
CAGTCCGTTTTGACGCAACCCCCGTCACTGAGTGCTGCGCCTGGGCAGAC

CATAACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTAATTAT

GTATCATGGTTCCAGCAATTCCCTGGCACGGCACCTCGCGTACTGATCTA

CGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAGC

AAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGTG

ATGAAGCTGATTACTACTGCGCTACTTGGGATAGCTCTCTTTCAGCTTAC

GTGTTTGGTTCCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 212)

KPS30
CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTC

AGTCGTCATCTCCTGCACTGGAACCAGCAGTGACATTGGGGCTAATAAC

TATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCTTGCT

TTATGAGGTCAATAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGCCT

CCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCTGGCTGAG

GATGAGGCTGATTATTACTGCTGCGGATATGGAGGCGGGAGGGTGTTCG

GCGGAGGGACCAAGCTGACCGTCCTAC (SEQ ID NO: 164)

KPD1
GAAATTGTGTTGACGCAGTCTCCAGGCATCCTGTCTTTGTCTCCAGGGGA

GAGAGCCACCCTCTCTTGCAGGGTCAGTCAGATTCTTTACATGTCTCATT

TGGCCTGGTATCAGCATAAACCTGGACAGGCTCCCAGACTCCTCATCTAT

GGTGCGTCCATCAGGGCCACTGGCGTCCCAGACAGGTTCAGTGGCAGTG

GGTCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGA

TTTTGCAGTGTATTACTGTCAGCAGTATGGCGCCTCACCGACGTTCGGCC

AAGGGACAATGGTGGAAATCAAAC (SEQ ID NO: 165)

KPL36
CAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAATC

AGTCACCATCTCCTGCACTGGAACCAGTAGTGACGTAGGTGGTAATAAC

TTTGTCTCCTGGTACCAACAGTATCCAGGCAAAGCCCCCAAACTCATTAT

TTATGAGGTCAATAAGCGGCCCTCAGGGGTCCCTGATCGTTTCTCTGGCT

CCAAGTCTGGCAACACGGCCTCCCTGACCGTCTCTGGGCTCCAGGCTGA

GGATGAGGCTGATTATTACTGCGGCGCATTTGGAGGTAGCAAGATGTTC

GGCGGAGGGACCAAGCTGACCGTCCTAG (SEQ ID NO: 198)

KPS44-G4
CAGTCCGTTTTGACGCAACCCCCGTCAGTGAGTGCTGCGCCTGGGCAGA

AGGTGACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATGC

TGTATCATGGTTCCAGCAACTGCCTGGCACGGCACCTAAACTGCTGATCT

ACGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAG

CAAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGT

GATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTA

CGTGTTTGGTACCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 222)

KPS44-G6
CAGTCCGTTTTGACGCAACCCCCGTCAGTGAGTGCTGCGCCTGGGCAGA

AGGTGACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGAGTC

TGTATCATGGTTCCAGCAACTGCCTGGCACGGCACCTAAACTGCTGATCT

ACGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAG

CAAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGT

GATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTA

CGTGTTTGGTACCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 232)

KPS44-G8
CAGTCCGTTTTGACGCAACCCCCGTCAGTGAGTGCTGCGCCTGGGCAGA

AGGTGACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATTC

TGTATCATGGTTCCAGCAACTGCCTGGCACGGCACCTAAACTGCTGATCT

ACGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAG

CAAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGT

GATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTA

CGTGTTTGGTACCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 242)

KPS44-G10
CAGTCCGTTTTGACGCAACCCCCGTCACTGAGTGCTGCGCCTGGGCAGAC

CATAACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATTCT

GTATCATGGTTCCAGCAATTCCCTGGCACGGCACCTCGCGTACTGATCTA

CGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAGC

AAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGTG

ATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTAC

GTGTTTGGTTCCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 252)

KPS44-G11
CAGTCCGTTTTGACGCAACCCCCGTCAGTGAGTGCTGCGCCTGGGCAGA

AGGTGACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATTC

TGTATCATGGTTCCAGCAACTGCCTGGCACGGCACCTAAACTGCTGATCT

ACGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAG

CAAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGT

GATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTA

CGTGTTTGGTACCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 262)

KPS44-G14
CAGTCCGTTTTGACGCAACCCCCGTCACTGAGTGCTGCGCCTGGGCAGAC

CATAACGATCGCCTGCTCAGGGACCAGCAGTAATATAGGCTCTGATTCT

GTATCATGGTTCCAGCAATTCCCTGGCACGGCACCTCGCGTACTGATCTA

CGAAAATAATAAGCGGCCCTCAGGCATTTCAGATAGGTTCTCTGGGAGC

AAGAGTGGTACAAGCGTAACGCTCGGTATCACCGGTCTCCAGACAGGTG

ATGAAGCTGATTACTACTGCGCTACTTTTGAGAGCTCTCTTTCAGCTTAC

GTGTTTGGTTCCGGGACCAAAGTGACAGTCCTC (SEQ ID NO: 272)

Also provided is a polynucleotide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to any one of the SEQ ID NOs provided in Table 5 or 6. Thus, in certain embodiments, the polynucleotide comprises (a) a polynucleotide having at least about 95% sequence identity to any one of the SEQ ID NOs provided in Table 5, and/or (b) a polynucleotide having at least about 95% sequence identity to any one of the SEQ ID NOs provided in Table 6. In certain embodiments, the polynucleotide comprises: (a) a polynucleotide having the sequence of a SEQ ID NO provided in Table 5; and/or (b) a polynucleotide having the sequence of a SEQ ID NO provided in Table 6.

The present disclosure provides an isolated polynucleotide or cDNA molecule sufficient for use as a hybridization probe, PCR primer or sequencing primer that is a fragment of a nucleic acid molecule disclosed herein or its complement. The nucleic acid molecule can, for example, be operably linked to a control sequence.

The present disclosure also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as described above (see, e.g., Tables 5 and 6).

The present disclosure also provides a recombinant host cell which comprises one or more nucleic acids, plasmids, vectors or as described above (see, e.g., Tables 5 and 6). A nucleic acid encoding any CDR or set of CDRs or VH domain or VL domain or antibody antigen-binding site, antibody, e.g., scFv, IgG1, or IgG2 as provided (see, e.g., Tables 1-4) itself forms an aspect of the present disclosure, as does a method of production of the encoded product, which method comprises expression from the nucleic acid encoding the product (e.g. the antigen binding protein, including, e.g., an anti-O2 antigen antibody or antigen-binding fragment thereof, disclosed herein). Expression can conveniently be achieved by culturing under appropriate conditions recombinant host cells containing a nucleic acid described herein. Following production by expression a CDR, set of CDRs, VH or VL domain, an antigen-binding protein can be isolated and/or purified using any suitable technique.

In some instances, the host cell is a mammalian host cell, such as a HEK293 cell, a HeLa cell, NS0 murine myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell.

Antigen-binding proteins, VH and/or VL domains and encoding nucleic acid molecules and vectors can be isolated and/or purified, e.g., from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes origin other than the sequence encoding a polypeptide with the required function. Nucleic acids according to the present disclosure can comprise DNA or RNA and can be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, yeast and baculovirus systems and transgenic plants and animals. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others. A common bacterial host is E. coli.

The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see for example Plückthun, A. Bio/Technology 9: 545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of an antigen-binding protein for example Chadd H E and Chamow S M (2001) 110 Current Opinion in Biotechnology 12: 188-194, Andersen D C and Krummen L (2002) Current Opinion in Biotechnology 13: 117, Larrick J W and Thomas D W (2001) Current opinion in Biotechnology 12:411-418.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acids, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1988, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 4^thedition 1999. The disclosures of Sambrook et al. and Ausubel et al. (both) are incorporated herein by reference.

Thus, a further aspect of the present disclosure provides a host cell containing nucleic acid as disclosed herein. For example, the disclosure provides a host cell transformed with nucleic acid comprising a nucleotide sequence (see, e.g., Tables 5 and 6) encoding an antigen-binding protein of the present disclosure or antibody CDR, set of CDRs, or VH and/or VL domain of an antigen-binding protein of the present disclosure (see, e.g., Tables 1-4). In some embodiments, the host cell comprises the expressed antigen-binding protein of the present disclosure or antibody CDR, set of CDRs, or VH and/or VL domain of an antigen-binding protein of the present disclosure (see, e.g., Tables 1-4).

Such a host cell can be in vitro and can be in culture. Such a host cell can be an isolated host cell. Such a host cell can be in vivo.

A still further aspect provided herein is a method comprising introducing such nucleic acid into a host cell. The introduction can employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g., vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell can use a viral or a plasmid based system. The plasmid system can be maintained episomally or may incorporated into the host cell or into an artificial chromosome. Incorporation can be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.

The introduction can be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the gene.

In one embodiment, the nucleic acid of the present disclosure is integrated into the genome (e.g. chromosome) of the host cell. Integration can be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

The present disclosure also provides a method which comprises using a construct (e.g. plasmid, vector, etc. as described above) in an expression system in order to express an antigen-binding protein or polypeptide as described above.

In another aspect, the disclosure provides a hybridoma producing the antigen-binding protein (e.g. anti-O2 antigen antibodies or antigen binding fragments thereof) of the disclosure.

A yet further aspect of the disclosure provides a method of production of an antibody binding protein (e.g. anti-O2 antigen antibodies or antigen binding fragments thereof) of the disclosure, the method including causing expression from encoding nucleic acid. Such a method can comprise culturing host cells under conditions suitable for production of said antigen-binding protein.

In some embodiments, the method of production further comprises isolating and/or purifying the antigen binding protein (including anti-O2 antigen antibodies or antigen binding fragments thereof) produced from the host cell or hybridoma.

EXAMPLES

Materials and Methods

Unless otherwise stated, all K. pneumoniae isolates were purchased from America Type Culture Collection, Eurofin collection, or IHMA collection, and cultures were maintained in 2×YT media at 37° C. supplemented with antibiotics when appropriate.

All statistical analysis was performed in GraphPad Prism version 6. For comparing bacterial burden, anti-O2 antigen antibody treated animals were compared with human isotype control antibody treated animals by unpaired t test. Survival results were plotted as Kaplan-Meier curves and analyzed as Log-rank (Mental-Cox) tests.

Unless otherwise specified, all antibodies used in the examples were in the human IgG1 format.

Example 1: Expansion of O2 Serotype in Multi-Drug Resistant (MDR) Strains

Highly purified LPS was generated from Klebsiella pneumoniae LPS serotype reference strains (Staten Serum Institute), which do not express capsular polysaccharides. SDS-PAGE analysis followed by Silver Stain confirmed the purity of LPS. Human antibodies against O2 serotypes were then identified based on the reactivity of human B cells against the respective purified LPS. The anti-O2 LPS antibodies (such as KPD1) showed cross-reactivity with O1 LPS, likely due to the common D-galactan I subunit shared between O1 and O2 LPS. (FIG. 1A).

Western blots were performed on 709 Klebsiella clinical isolates collected between 2012 and 2014 from various geographical locations spanning six continents and various sites of infection. The KPD1 antibody was used to assess the propensity of O2 isolates. Contrary to historical literature demonstrating the propensity of O1 isolates (Trautmann, M, et. al. 2004. O antigen seroepidemiology of Klebsiella clinical isolates and implications for immunoprophylaxis of Klebsiella infections. Vaccine. 22: 818-821.), O2 was the most prevalent LPS serotype (35.8%, FIG. 1B). Isolates were further categorized into three different drug susceptibility groups to determine if the increased incidence of O2 may be attributed to an overall increase in multi-drug resistant (MDR) isolates. The susceptibility groups were based on minimum inhibitory concentration (MIC) information provided by IHMA. Extended spectrum beta-lactamase producing (ESBL) strains are resistant to ceftazidime, but susceptible to carbanpenem. Carbapenem-Resistant Enterobacteriaceae (CRE) strains are resistant to carbanpenems. Interestingly, steady increases were observed in the O2 prevalence in multi-drug resistant isolates. The increases of O2 serotype were even more pronounced in CRE strains, suggesting the unique expansion of O2 MDR strains (see FIG. 1B).

Example 2: Isolation of K. pneumoniae O2 Specific Hybridomas

BALB/c mice were immunized weekly with O2 lipopolysaccharide (LPS) via subcutaneous route for four weeks. At the end of the immunization, lymph node and splenic B cells were harvested and fused with P3X myelomas. Supernatants from the resulting hybridomas were then screened for binding to Klebsiella pneumoniae 43816DM by whole bacterial enzyme-linked immunosorbent assay (ELISA). Positive hybridomas were sub-cultured in antibiotic-free medium, and the supernatants were subjected to ELISA binding and opsonophagocytic killing (OPK) assay to select for potentially protective hybridomas against K. pneumoniae O2 LPS. The 6F6 antibody was obtained from this method.

Example 3: Isolation of K. pneumoniae O2 Specific Antibodies from Tonsil and Patient B Cells

Peripheral blood mononuclear cells (PBMC) and sera were separated from buffy coats from healthy blood donors or convalescent patients after K. pneumoniae infection as described in Beltramello M. et al., Cell Host Microbe. 8, 271-283 (2010). PMBC were stored in liquid nitrogen whereas the plasma was stored at 4° C. Alternatively lymphocytes were obtained from tonsils or adenoids after tissue homogenization in the presence of DNAaseI and collagenase. Memory B cells were isolated from cryopreserved PMBC or from lymphocytes isolated from tonsils or adenoids using CD19 microbeads, followed by depletion of cells carrying IgM, IgD, and IgA by cell sorting. Memory B cells were immortalized as described in Traggiai, E. et al., Nature Medicine 10: 871-875 (2004).

For PBMC donor selection the corresponding plasmas were diluted in PBS and used to determine the presence of antibodies binding to different pools of bacterial strains or to purified bacterial antigens (e.g. LPS or other polysaccharides, bacterial proteins) by ELISA.

For tonsil and adenoid donor selection, tonsillar lymphocytes were polyclonally stimulated as described in Pinna, D., et al., European Journal of Immunology 39: 1260-1270 (2009). Supernatants containing polyclonal antibody mixtures were used to determine the presence of antibodies binding to different pools of bacterial strains or to purified bacterial antigens (e.g. LPS or other polysaccharides, bacterial proteins) by enzyme-linked immunosorbent assay (ELISA).

The antibody KPD1 was isolated by screening B cells from peripheral blood mononuclear cells (PBMC) of a healthy donor in ELISA with plates coated with different K. pneumoniae strains. In secondary screenings, KPD1 showed binding to LPS-O1 and O2, neutralized LPS-O1 and O2, and showed OPK activity against an O2 capsule mutant. KPD1 was isolated as a human IgG2 antibody.

The antibodies KPS3, KPS24, KPS18, KPS30, and KPS44 were obtained by screening B cells of a convalescent donor in ELISA with plates coated with LPS-O2. In secondary screening KPS30 bound LPS-O1 and-O2, whereas KPS3, KPS18 and KPS24 bound specifically to LPS-O2. KPS3, 18, 24, 30, and 44 showed strong OPK activity against an O2 capsule mutant strain, but not the O1 capsule mutant strain. KPS3, KPS24, KPS18 neutralized LPSO2, KPS30 neutralized both LPS-O1 and LPS-O2. KPS3, KPS18, KPS24, KPS30 and KPS44 were isolated as human IgG2 antibodies.

The antibodies KPN17, KPN42, KPN44, KPN70, and KPN179 were isolated by interrogating sorted IgG2 B cells from tonsil 21 in ELISA with LPS-O2. KPN17, KPN42, KPN44 and KPN179 were shown to bind LPS-O1 and-O2 in secondary screening by ELISA. KPN17 and KPN42 neutralize LPS-O2, KPN44 and KPN70 neutralize both LPS-O2 and LPS-01. KPN179 did not show LPS-neutralizing activity. KPN17, KPN42 and KPN44 showed OPK activity against an O2 capsule mutant.

The antibodies KPL26 and KPL36 were isolated by interrogating sorted IgG2 B cells from tonsil 14 in high content flow cytometry for binding to O2 strains. In secondary screening they were confirmed to bind to LPS-O1 and LPS-O2 in ELISA and KPL26 to neutralize LPS-O1 and LPS-O2, KPL36 to neutralize LPS-O2. KPL26 showed OPK activity against O1 and O2 capsule mutants.

Example 4: Summary of Three Classes of O2 LPS mAbs

As described in more detail in below, anti-O2 LPS mAbs were tested for: 1) binding to O1 and O2 LPS (see Example 5); 2) LPS neutralization (LPS-Neut) against O1 and O2 LPS (see Example 7); 3) OPK against capsule mutant strain 43816AcpsB lux (01) and 8570ΔcpsB lux (O2) (see Example 8); and 4) protective activity in pneumonia models (see Example 9). O2 LPS mAbs were subsequently divided into three classes based on their in vitro activities. Class I mAbs were characterized as Binding (O1+O2+)/OPK (O1−O2+); Class 2 mAbs were characterized as Binding (O1+O2+)/OPK (O1+O2+); and Class III mAbs were characterized as Binding (O1±O2+)/OPK (O1−O2+). Table 7 summarizes the binding characteristics of the three classes of Anti-O2 LPS mAbs.

TABLE 7

Summary of Anti-O2 Antigen Antibodies.

Mouse

Original
LPS
LPS Neut
D Capsule OPK
protection

Catagory
mAb
Isotype
binding
O1
O2
O1
O2
models

Class I
KPD1
Hu rIgG2
O1/O2
++
++
−
++
−
(O1/O2)

6F6
Ms IgM

NT
NT
−
+
−
(O2)

KPN17
Hu rIgG2

−
+/−
−
+
−
(O2)

KPN70
Hu rIgG2

++
++
−
+++
+/−
(O1/O2)

KPS30
Hu rIgG2

++
++
−
+++
NT

Class II
KPL26
Hu rIgG2
O1/O2
+
+
+
+
+
(O1)

KPL36
Hu rIgG2

−
+
+
+
NT

Class III
KPN42
Hu rIgG2
O2,
−
+/−
−
+++
+++
(O2)

KPN179
Hu rIgG2
weak O1
−
+/−
−
+++
+++
(O2)

KPS3
Hu rIgG2

−
+/−
−
+++
NT

KPS18
Hu rIgG2

−
+/−
−
+++
NT

KPS24
Hu rIgG2

−
+/−
−
+++
NT

KPS44v2017
Hu rIgG2

−
+/−
−
+++
+++
(O2)

KPS44
Hu rIgG2

−
+/−
−
+++
+++
(O2)

NT: not tested

+++: Strongly positive activity

+: Positive activity

+/−: Somewhat positive depending upon the test conditions

Notably, significant LPS neutralization activity is not required for high levels of in vivo protection.

Example 5: Enzyme-Linked Immunosorbent Assay (ELISA)

For screening by ELISA, spectraplate-384 with high protein binding treatment (custom made from Perkin Elmer, CUSG83093) were coated overnight at 4° C. with 5 μg/ml O1 or O2 LPS in phosphate-buffered saline (PBS), pH 7.2, and plates were subsequently blocked with PBS-B, i.e. PBS supplemented with 1% endotoxin free BSA (Sigma, #A9430). The coated plates were incubated with cell culture supernatants from polyclonally stimulated lymphocytes (AMBRA) or from monoclonal immortalized B cells (donor interrogations) containing fully human antibodies or with diluted plasma samples (PB MC donor selections) for 1 hour at room temperature. The plates were then washed with PBS containing 0.1% Tween-20 (PBS-T). Secondary antibody was added; either Alkaline Phosphatase-conjugated Goat Anti-Human IgG-AP (Southern Biotech, 2040-04, 1:1000 in PBS-B) or Peroxidase AffiniPure F(ab′)₂Fragment Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch #309 036 098, 1:5000 in PBS-B) were used. Secondary antibodies were typically incubated for 1 h. Plates were washed three times with PBS-T, and P-NitroPhenyl Phosphate (pNPP, Sigma-Aldrich, cat #N2765-100TAB) or Sureblue (KPL, 53-00-03) substrates were added and incubated for 10 min or until the development of a colorimetric reaction. In the case of the HRP substrate Sureblue, the reaction was stopped by adding an equal volume of 0.2N HCl. The absorbance at 405 nm (pNPP) or 450 nm (Sureblue) was measured by a microplate reader (Biotek, Elx808).

To determine binding EC50 values ELISA was performed in 96 well plates (Maxi sorp, Nunc #442404) coated overnight at 4° C. with 5 μg/ml O1 or O2 LPS in phosphate-buffered saline (PBS), pH 7.2, and plates were subsequently blocked with PBS-B, i.e. PBS supplemented with 1% endotoxin free BSA (Sigma, #A9430). The coated plates were incubated with serial dilutions of the monoclonal antibodies for 1 hour at room temperature. The plates were then washed with PBS containing 0.1% Tween-20 (PBS-T). Alkaline Phosphatase-conjugated Goat Anti-Human IgG (Southern Biotech, 2040-04, 1:1000 in PBS-B) was added. The secondary antibody reactions were incubated for 1 h. Plates were washed three times with PBS-T, and P-NitroPhenyl Phosphate (pNPP, Sigma-Aldrich, cat #N2765-100TAB) substrate was added and incubated for 30 min or until the development of a colorimetric reaction. The absorbance at 405 nm was measured by a microplate reader (Biotek, Elx808). The data was plotted with Graphpad Prism software. Representative mAbs from each class were tested for their binding to O1 LPS (FIG. 2A) and O2 LPS (FIG. 2B), and EC50 values were obtained (FIG. 2C). The majority of the anti-O2 LPS mAbs bound to both O1 and O2 LPS by ELISA. Among these, KPN42 showed lower affinity to O1 than O2. None of these LPS mAbs bound to the O3, O4, O5, O7, or O12 LPS serotypes (see e.g., FIG. 1A).

Example 6: Octet Binding Assay with Anti-O2 mAbs

The interaction of anti-O2 LPS mAbs with O1 and O2 LPS was further tested in solution phase by Octet platform. This platform provides a powerful tool to measure the rate of biomolecular complex formation and complex stability in a more biologically meaningful setting. Briefly, Protein A coated sensors were coated with 0.2 μg/mL anti-O2 LPS mAbs for 10 minutes before being dipped into solution containing 2 μg/mL O1 or O2 LPS in Kinetics buffer (ForteBio, dilute 10× to 1× with PBS). Changes in the number of molecules bound to the biosensor caused a shift in the interference pattern that was recorded in real time. As shown in FIGS. 3A and 3B, Class I and Class II mAbs bound to both O1 and O2 LPS, while Class III mAb (KPN42) showed no binding to O1 LPS. The affinity constant (K_D) of Class III mAbs to O2 LPS was calculated based on the on-rate and off-rate from Octet sensorgram. Both KPN42 and KPN179 showed comparable affinity constant at the average of 4.8E-09 and 7.98E-09M, respectively (FIG. 3C).

Example 7: LPS Neutralization Assays

Activation of TLR4 receptors by bacterial LPS leads to downstream activation of the NF-κB transcriptional regulator. A decrease in induction of NF-κB-responsive luciferase activity was used to quantify LPS neutralization activity by LPS mAbs. A murine RAW264.7 macrophage cell line was engineered to carry a firefly luciferase reporter gene under the control of an NF-κB-responsive promoter (RAW264.7-lux). Serially diluted antibody stocks were mixed with LPS in a 1:1 ratio and incubated at 4° C. for 1 hr. Antibody/LPS mixtures were then diluted 1:10 into assay plates containing pre-seeded RAW264.7-lux cells (4e5 cells/well), which were then placed at 37° C. with 5% CO2 for 2.5 hours. Following incubations, Steady Glo solution (Promega) was added to each well and incubated for another 20 min protected from light. The relative light units (RLUs) were measured using a multi-mode microplate reader (Synergy 2, Biotek or envision multilabel plate reader, Perkin Elmer). The percentage of inhibition was determined by comparing RLU derived from assays with no antibodies to RLU derived from assays with anti-K. pneumoniae mAbs and assays performed with a negative control mAb. Results from these neutralization assays are shown in FIG. 4. The KPD1, KPN44, KPN70, and KPL26 blocked O1 and O2 LPS activation of NF-κB, while KPN42, KPN17, KPL36, KPS3, KPS18, KPS24, and KPS44 showed moderate neutralizing activity against O2 LPS, but no activity against O1 LPS. All mAbs were tested in human IgG1 format.

Example 8: OPK Activity of Selected O2 mAbs

OPK activity of anti-O2 LPS mAbs was tested against O1 and O2 strains. Briefly, log phase cultures of luminescent K. pneumoniae strains 8570ΔcpsBLux (O2) and 43816ΔcpsBLux (01) were diluted to ˜2×10⁶cells/ml. K. pneumoniae cells, 5e5/well dimethylformamide (DMF) differentiated HL-60 cells, cleared baby rabbit serum (1:10 Cedarlane), and a series dilution of antibodies (2 ng-2.5 μg/mL) were mixed in 96-well plates and incubated at 37° C. for two hours with shaking (250 rpm). The relative light units (RLUs) were then measured using a multi-mode microplate reader (Synergy 2, Biotek or envision multilabel plate reader, Perkin Elmer). The percentage of killing was determined by comparing RLU derived from assays with no antibodies to RLU derived from assays with anti-K. pneumoniae mAbs and assays performed with a negative control mAb. Positive killing was determined where the percentage of killing is above 40%. All anti-O2 IgG1 mAbs tested showed strong OPK activity (80-100% killing) against O2 capsule mutant strains (FIG. 5 and FIG. 10). KPL26 induced OPK activity to both O1 and O2 strains (FIG. 5).

Example 9: Class III mAbs Protect in Lethal Pneumonia Models

C57/BL6 mice were obtained from Jackson Laboratories and maintained in a special pathogen-free facility. All animal experiments were conducted in accordance with Institutional Animal Care and Use Committee (IACUC) protocol and guidance. K. pneumoniae strains were grown on agar plates overnight and diluted in saline at proper concentration. The inoculum titer was determined by plating a serial dilution of bacteria onto agar plates prior to and post challenge. In acute pneumonia models, C57/BL6 mice were inoculated with 1e4 to 2e8 colony-forming units (CFU) of K. pneumoniae clinical isolates in 50 μl saline intranasally. Anti-K. pneumoniae monoclonal antibodies (mAbs) and human IgG1 control antibodies were given 1-24 hour post bacterial challenge (therapeutic dosing). Mouse survival was monitored daily until up to day 8. Survival data of representative experiments were plotted in Graphpad Prism software.

Mice were challenged with 2e8 CFU of Klebsiella pneumoniae Carbanpenemase (KPC) strain Kp961842_O2 or with 3e8 CFU of KPC strain Kp977778_O2, followed by the administration of anti-O2 mAbs. When administered at a concentration of 2 mg/kg 1 hour post bacterial infection, Class III anti-O2 LPS mAbs KPN42, KPN179-IgG1, and KPS44v2017 protected mice from lethal bacterial challenge with the multi-drug resistant KPC strains Kp961842_O2 (FIG. 6, left panel, and FIG. 10) and Kp977778_O2 (FIG. 6B, right panel). KPN179-IgG3 also conferred protection, while the Class I mAb KPN70-hIgG1 or KPD1-IgG1 did not protect in these two models. Class II mAb KPL26 conferred moderate protection in a lethal O1 pneumonia model, while did not show protection in Kp961842_O2 pneumonia model.

Example 10: Anti-O2 LPS mAbs KPN42 and KPN179 Show Strong Synergy with Antibiotic Meropenem in Lethal Pneumonia Model

The lethal pneumonia model as described in Example 9 was used to assess the activity of anti-O2 LPS antibodies in combination with an antibiotic. Both antibiotic and antibody were administered 1 hour after bacterial infection. The combination of meropenem human equivalent dosage (50 mpk) with sub-therapeutic dosages (0.2 mpk) of KPN42 or KPN179 showed significantly improved protection compared to antibody or antibiotic monotherapy (FIG. 7). These results illustrate that administration of an anti-O2 antibody, including KPN42 or KPN179, sensitizes antibiotic resistant Klebsiella pneumoniae strains to antibiotic therapy and that sub-therapeutic doses of the anti-O2 antibodies KPN42 or KPN179 show strong synergy with antibiotics in antibiotic resistant Klebsiella pneumoniae strains.

Example 11: KPN42 Protects Up to 6 Hour Post Infection in Conjunction with Meropenem

50 mg/kg of meropenem and 2 mg/kg of antibody were administered at 1, 2, 4, 6, and 24 hour after bacterial infection, as described for the antibody and antibiotic combination studies in Examples 9 and 10. The combination of meropenem and KPN42 showed significantly better protection than a combination of a control mAb and meropenem up to 6 hours post infection (FIG. 8). The combination of KPN179 and meropenem conferred better protection than a combination of a control mAb and meropenem up to 4 hours post infection (FIG. 8). Together with Example 10, these results illustrate the efficacy of administering an anti-O2 antibody, including KPN42 or KPN179, after bacterial infection (i.e., therapeutic administration).

Example 12: KPN42 and KPN179 Sequence Optimization

In order to reduce sequence liability for mAb development and potential anti-drug antibody, an unpaired cysteine in the light chain CDR3 of KPN42 was exchanged with alanine (see FIG. 9, antibody A (KPN42-FR-GL-VH/KPN42-FR-GL-C105A-VL) and antibody B (KPN42-FR-1-2-4-GL-VH/KPN42-FR-GL-C105A-VL)), and somatic mutations in the frameworks of KPN42 (see FIG. 9, antibody A) or in the frameworks 1, 2, and 4 of KPN42 (see FIG. 9, antibody B) were replace with germline residues. Similarly an unpaired cysteine in the light chain CDR3 of KPN179 was exchanged with alanine, and an asparagine residue forming a deamidation motif in the KPN179 heavy chain CDR1 was replaced with the germline residue serine (see FIG. 9, antibody C (KPN179-FR-GL-N35S-VH/KPN179-FR-GL-C105A-VL) and antibody D (KPN179-FR-1-2-4-GL-N35S-VH/KPN179-FR-GL-C105A-VL)), somatic mutations in the frameworks of KPN179 (see FIG. 9, antibody C) or in frameworks 1, 2 and 4 of KPN179 were replaced with germline residues (see FIG. 9, antibody D). Replacement of all somatic mutations in KPN179 VH and VL frameworks with germline residues (see FIG. 9, antibody C) significantly reduced binding to O2 LPS. The rest of the mutations showed comparable binding to parent antibodies (see, e.g., FIG. 9, antibodies A, B and D).

Example 13: Anti-O2 Antigen Antibodies Bind to Clinically Relevant Klebsiella

In order to determine if anti-O2 antigen antibodies bind to clinically relevant Klebsiella strains, the binding of anti-O2 mAbs to clinical isolates was determined by western blot assay. Briefly, purified LPS or bacterial lysates were subjected to sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE). Separated proteins and LPS were transferred from gels to nitrocellulose membranes with an iBlot apparatus based on the manufacturer's recommendation. (Life Technology). Membranes were then blocked with Casein blocking buffer before being probed with O2 (KPD1) monoclonal antibodies or antibodies specific for other LPS serotypes. After three washes with 0.05% Tween in PBS buffer (PBS-T), blots were incubated with IRDye680 or 800 fluorescent 2^ndantibodies (Licor). Blots were visualized with an Odyssey Image Station. Distinct laddering patterns were observed for LPS blots. Assays were repeated at least twice. In some circumstances, bacterial lysates were treated with Protease K to remove protein components before the western blot analysis. Strains that bind to KPD1, but not to other LPS serotype specific mAbs were characterized as O2 strains. A summary of the clinically relevant Klebsiella strains to which anti-O2 mAbs KPD1 and KPN42 bind is shown in Table 8.

TABLE 8

Summary of clinical Klebsiella strains to which anti-O2 mAbs bind.

IHMA
Body
Facility
Molecular

Region
Country
Number
Location
Name
Summary

1
Latin
Chile
847204
INT: Wound
Surgery
SHV-11(b); TEM-1(b);

America

General
KPC-2;

2
Latin
Argentina
847378
Respiratory:
Medicine ICU
KPC-2;

America

Endotracheal aspirate

3
Latin
Argentina
847383
Respiratory:
Medicine ICU
SHV-11(b); TEM-1(b);

America

Bronchoalveolar

CTX-M-15; NDM-1;

lavage

4
Latin
Argentina
847387
Respiratory:
Medicine ICU
SHV-11(b); TEM-1(b);

America

Bronchoalveolar

CTX-M-15; NDM-1;

lavage

5
Latin
Argentina
847694
Unknown
Medicine ICU
SHV-11(b); KPC-2;

America

6
Latin
Argentina
847747
Respiratory:
Medicine ICU
SHV-11(b); KPC-2;

America

Endotracheal aspirate

7
Europe
Greece
848832
Respiratory: Sputum
General
SHV-1(b); CTX-M-15;

Unspecified
KPC-2;

ICU

8
Europe
Greece
848844
Respiratory: Sputum
General
SHV-12(e); KPC-2;

Unspecified

ICU

9
Europe
Greece
848876
Respiratory:
Medicine
SHV-1(b); TEM-1(b); CTX-

Bronchial brushing
General
M-2;

10
Middle
Israel
849156
Bodily Fluids:
Medicine
SHV-1(b); TEM-1(b); CTX-

East

Peritoneal
General
M-2;

11
Middle
Israel
849584
INT: Abscess
Pediatric ICU
SHV- 11(b); TEM- 1(b);

East

KPC-3;

12
Middle
Israel
849585
INT: Wound
Medicine
SHV-1(b); KPC-3;

East

General

13
North
United
854022
Respiratory:
General
SHV-11(b); TEM-1(b);

America
States

Endotracheal aspirate
Unspecified
CTX-M-15; OXA-48(c)

ICU

14
North
United
857973
Respiratory:
Other
SHV-11(b); TEM-1(b);

America
States

Endotracheal aspirate

CTX-M-15; OXA-48(c)

15
North
United
857978
Respiratory:
Medicine
SHV-11(b); TEM-1(b);

America
States

Endotracheal aspirate
General
CTX-M-15;

16
North
United
863890
INT: Decubitus
None Given
SHV-11(b); TEM-1(b);

America
States

KPC-2;

17
North
United
863930
Bodily Fluids:
Surgery ICU
SHV-11(b); TEM-1(b);

America
States

Peritoneal

KPC-2;

18
Europe
Italy
867822
Bodily Fluids:
Surgery
SHV-11(b); TEM-1(b);

Peritoneal
General
KPC-3;

19
Middle
Israel
869311
Respiratory:
Medicine ICU
SHV-11(b); KPC-3;

East

Bronchial brushing

20
Europe
Romania
869918
Respiratory: Sputum
General
SHV-11(b); TEM-1(b);

Unspecified
KPC-2;

ICU

21
Europe
Russia
874316
Respiratory: Sputum
General
SHV-11(b); CTX-M-55;

Unspecified
OXA-48(c)

ICU

22
Europe
Russia
874329
Respiratory: Other
General

Unspecified

ICU

23
Europe
Russia
874876
Respiratory: Sputum
Pediatric ICU
SHV-11(b); TEM-1(b);

CTX-M-15; KPC-2;

24
Europe
Belgium
875655
Respiratory:
General
SHV-28(e); TEM-1(b);

Endotracheal aspirate
Unspecified
CTX-M-15; KPC-3;

ICU

25
Europe
Italy
875926
Respiratory: Sputum
Medicine
SHV-11(b); KPC-3;

General

26
Europe
Italy
875928
Respiratory:
Medicine ICU
SHV-2(e);

Endotracheal aspirate

27
Europe
Italy
875931
Respiratory:
Medicine
SHV-11(b); KPC-3;

Bronchoalveolar
General

lavage

28
Latin
Brazil
900678
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America

Endotracheal aspirate

OSBL(u); VEB-2; KPC-2;

29
Europe
Italy
918904
Respiratory:
Medicine
SHV-OSBL(u); TEM-

Bronchoalveolar
General
OSBL(u); KPC-3;

lavage

30
Europe
Greece
921185
Respiratory: Sputum
Medicine
SHV-12(e); KPC-2;

General

31
Europe
Turkey
926871
Respiratory:
General
SHV-OSBL(u); CTX-M-15;

Endotracheal aspirate
Unspecified
OXA-48(c)

ICU

32
Europe
Turkey
926901
Respiratory: Sputum
Medicine
SHV-OSBL(u); CTX-M-15;

General
OXA-48(c)

33
Europe
Greece
927850
Respiratory:
Medicine ICU
SHV-OSBL(u); OXA-48(c)

Endotracheal aspirate

34
Europe
Greece
927897
Respiratory:
General
SHV-OSBL(u); TEM-

Endotracheal aspirate
Unspecified
OSBL(u); CTX-M-2;

ICU

35
Europe
Greece
927898
Respiratory:
General
SHV-OSBL(u); KPC-2;

Endotracheal aspirate
Unspecified

ICU

36
Europe
Greece
927915
Respiratory:
General

Endotracheal aspirate
Unspecified

ICU

37
Europe
Greece
927952
Respiratory:
General

Endotracheal aspirate
Unspecified

ICU

38
Europe
Greece
927963
Respiratory:
General

Endotracheal aspirate
Unspecified

ICU

39
Europe
Greece
927964
Respiratory:
General
TEM-OSBL(u); CTX-M-

Endotracheal aspirate
Unspecified
15; CTX-M-27; NDM-1;

ICU

40
Middle
Israel
937433
Respiratory: Sputum
Medicine
SHV-OSBL(u); KPC-3;

East

General

41
Europe
Romania
938940
INT: Wound
Medicine
SHV-OSBL(u); TEM-

General
OSBL(u); KPC-2;

42
Europe
Romania
939003
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Endotracheal aspirate

OSBL(u); KPC-2;

43
Latin
Argentina
939866
Respiratory: Lungs
Surgery ICU
SHV-OSBL(u); TEM-

America

OSBL(u); CTX-M-15;

OXA-48(c)

44
Latin
Argentina
939929
Respiratory:
General
SHV-12(e); TEM-OSBL(u);

America

Bronchial brushing
Unspecified
KPC-2;

ICU

45
Latin
Argentina
939943
Respiratory:
General
SHV-12(e); TEM-OSBL(u);

America

Bronchial brushing
Unspecified
KPC-2;

ICU

46
Europe
Italy
946889
Respiratory:
General
SHV-OSBL(u); TEM-

Bronchoalveolar
Unspecified
OSBL(u); KPC-2;

lavage
ICU

47
Europe
Italy
946897
Respiratory:
Medicine
SHV-OSBL(u); KPC-3;

Bronchial brushing
General

48
Europe
Italy
946900
Respiratory:
Surgery
SHV-OSBL(u); TEM-

Bronchial brushing
General
OSBL(u); KPC-3;

49
Europe
Italy
947475
Respiratory: Lungs
Medicine
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-65;

KPC-2;

50
Latin
Colombia
960228
INT: Wound
Medicine
SHV-OSBL(u); TEM-

America

General
OSBL(u); KPC-2;

51
Latin
Colombia
960249
Respiratory: Other
Medicine
SHV-OSBL(u); KPC-2;

America

General

52
North
United
961842
Respiratory:
Medicine ICU
SHV-12(e); TEM-OSBL(u);

America
States

Bronchoalveolar

CTX-M-65; KPC-2;

lavage

53
Africa
South
963278
Respiratory:
Medicine ICU
SHV-12(e); KPC-2;

Africa

Endotracheal aspirate

54
South
Philippines
966426
Respiratory: Sputum
Medicine
SHV-12(e); TEM-OSBL(u);

Pacific

General
CTX-M-15; NDM-7;

55
Europe
Italy
971222
Respiratory:
Medicine
SHV-OSBL(u); TEM-

Bronchial brushing
General
OSBL(u); KPC-3;

56
Europe
Russia
975977
Respiratory:
Surgery ICU
SHV-12(e); TEM-OSBL(u);

Endotracheal aspirate

KPC-2;

57
Europe
Russia
976037
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

Endotracheal aspirate

OSBL(u); CTX-M-15;

OXA-244(c)

58
Europe
Russia
976078
Respiratory:
Surgery ICU
SHV-12(e); TEM-OSBL(u);

Endotracheal aspirate

KPC-2;

59
Latin
Argentina
977128
INT: Wound
Medicine
SHV-12(e); TEM-OSBL(u);

America

General
KPC-2;

60
Latin
Argentina
977314
Bodily Fluids:
Medicine
SHV-12(e); KPC-2;

America

Peritoneal
General

61
North
United
977778
GI: Abscess
Medicine
SHV-OSBL(u); KPC-3;

America
States

General

62
North
United
978960
INT: Wound
Medicine
SHV-12(e); TEM-OSBL(u);

America
States

General
KPC-3;

63
North
United
978971
Respiratory: Sputum
Medicine ICU
SHV-OSBL(u); TEM-

America
States

OSBL(u); KPC-3;

64
North
United
979049
Respiratory: Sputum
Medicine
CTX-M-12; KPC-2;

America
States

General

65
North
United
979288
Respiratory: Sputum
Surgery
SHV-OSBL(u); KPC-3;

America
States

General

66
North
United
979290
Respiratory: Sputum
Medicine ICU
SHV-12(e); TEM-OSBL(u);

America
States

KPC-3;

67
Latin
Brazil
990976
Bodily Fluids:
None Given
SHV-OSBL(u); CTX-M-2;

America

Peritoneal

KPC-2;

68
Latin
Brazil
991499
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America

Endotracheal aspirate

OSBL(u); CTX-M-15;

NDM-1;

69
Middle
Israel
994039
Respiratory:
Medicine
SHV-OSBL(u); CTX-M-15;

East

Endotracheal aspirate
General
NDM-1;

70
Asia
China
995976
Respiratory: Sputum
Pediatric ICU
SHV-OSBL(u); TEM-

OSBL(u); CTX-M-15;

OXA-48(c)

71
Europe
Greece
1013421
Respiratory: Sputum
Medicine

General

72
Europe
Greece
1013442
INT: Skin Ulcer
General
SHV-OSBL(u); CTX-M-15;

Unspecified
OXA-48(c)

ICU

73
Africa
Nigeria
1043242
INT: Wound
Medicine
SHV-55(e); TEM-OSBL(u);

General
CTX-M-15; NDM-1;

74
Europe
Russia
1049163
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Endotracheal aspirate

OSBL(u); CTX-M-15;

75
Europe
Russia
1049391
Respiratory:
Surgery ICU
SHV-12(e); TEM-OSBL(u);

Bronchoalveolar

KPC-2;

lavage

76
Europe
Russia
1049400
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

77
Europe
Russia
1049474
Respiratory:
Surgery
SHV-12(e); TEM-OSBL(u);

Bronchoalveolar
General
KPC-2;

lavage

78
Europe
Russia
1049592
Respiratory: Sputum
Medicine ICU
SHV-OSBL(u); CTX-M-3;

OXA-48(c)

79
Europe
Spain
1073956
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Bronchial brushing

OSBL(u); KPC-3;

80
South
Philippines
1079540
CVS: Blood
Pediatric ICU
SHV-12(e); TEM-OSBL(u);

Pacific

KPC-3;

81
South
Philippines
1079544
Respiratory:
Medicine ICU
TEM-OSBL(u); KPC-3;

Pacific

Endotracheal aspirate

82
Europe
Italy
1081144
Respiratory: Sputum
Medicine ICU

83
Europe
Greece
1081949
CVS: Blood
Medicine

General

84
Europe
Greece
1081956
CVS: Blood
Surgery
SHV-OSBL(u); TEM-

General
OSBL(u); KPC-3;

85
Europe
Greece
1081997
Respiratory:
General
SHV-OSBL(u); TEM-

Bronchoalveolar
Unspecified
OSBL(u); KPC-3;

lavage
ICU

86
Europe
Greece
1082051
Respiratory:
General
SHV-12(e); TEM-OSBL(u);

Bronchoalveolar
Unspecified
KPC-2;

lavage
ICU

87
Europe
Greece
1082058
CVS: Blood
Other

88
Europe
Greece
1082074
CVS: Blood
General

Unspecified

ICU

89
Europe
Greece
1082098
CVS: Blood
General

Unspecified

ICU

90
Asia
Korea,
1085601
Respiratory: Sputum
Medicine
SHV-12(e); TEM-OSBL(u);

South

General
KPC-2;

91
Europe
Hungary
1090072
Bodily Fluids:
Surgery

Peritoneal
General

92
Africa
South
1093894
Bodily Fluids:
General
SHV-OSBL(u); CTX-M-15;

Africa

Peritoneal
Unspecified

ICU

93
Latin
Argentina
1093960
Respiratory:
Medicine ICU
SHV-OSBL(u); CTX-M-15;

America

Bronchoalveolar

KPC-2;

lavage

94
Latin
Argentina
1093976
Respiratory:
Medicine ICU
SHV-OSBL(u); KPC-2;

America

Bronchoalveolar

lavage

95
Latin
Argentina
1093980
Bodily Fluids:
Emergency

America

Peritoneal
Room

96
North
United
1105534
Respiratory:
Medicine
SHV-OSBL(u); TEM-

America
States

Bronchoalveolar
General
OSBL(u); KPC-2;

lavage

97
North
United
1105547
Respiratory:
Medicine

America
States

Bronchoalveolar
General

lavage

98
Latin
Colombia
1109216
Bodily Fluids:
Surgery
SHV-OSBL(u); KPC-3;

America

Peritoneal
General

99
Europe
Czech
1120042
Respiratory: Sputum
Medicine ICU
SHV-OSBL(u); TEM-

Republic

OSBL(u); CTX-M-15;

100
Europe
Austria
1127552
INT: Wound
Medicine ICU

101
Europe
Italy
1137983
GI: Abscess
Surgery
SHV-OSBL(u);

General

102
Europe
Italy
1137984
Respiratory:
Medicine ICU
SHV-12(e); TEM-OSBL(u);

Bronchial brushing

KPC-2;

103
Europe
Italy
1137991
Bodily Fluids:
Surgery

Peritoneal
General

104
Latin
Chile
969740
INT: Wound
Surgery
SHV-OSBL(u); TEM-

America

General
OSBL(u); CTX-M-15;

105
Latin
Chile
969743
Respiratory:
Medicine
SHV-OSBL(u); TEM-

America

Endotracheal aspirate
General
OSBL(u); CTX-M-15;

CTX-M-2;

106
Latin
Argentina
977113
Respiratory:
Medicine ICU

America

Endotracheal aspirate

107
North
United
1094435
INT: Wound
Medicine

America
States

General

108
North
United
1147894
Respiratory:
Medicine ICU

America
States

Endotracheal aspirate

109
Europe
Italy
947566
GU: Urine
Medicine
SHV-12(e); VIM-New

General
V ariant;

110
Latin
Argentina
847700
GU: Urine
Medicine
TEM-1(b); KPC-2;

America

General

111
Latin
Argentina
847708
GU: Urine
Medicine ICU
SHV-11(b); TEM-1(b);

America

KPC-2;

112
Europe
Greece
848827
GU: Urine
Emergency
SHV-11(b); TEM-1(b);

Room
KPC-2;

113
Europe
Greece
848828
GU: Urine
General
SHV-11(b); TEM-1(b);

Unspecified
KPC-2;

ICU

114
Europe
Greece
848829
GU: Urine
Other
SHV-11(b); KPC-2;

115
Europe
Greece
848843
GU: Urine
Medicine
SHV-11(b); TEM-1(b);

General
KPC-2;

116
Middle
Israel
869317
GU: Urine
Other
SHV-11(b); TEM-1(b);

East

KPC-3;

117
North
United
872020
GU: Urine
Medicine ICU
SHV-28(e); TEM-1(b);

America
States

CTX-M-15; KPC-2;

118
Europe
Turkey
889939
GU: Urine
Surgery ICU
SHV-1(b); OXA-48(c)

119
North
United
897067
GU: Urine
Surgery ICU
SHV-11(b); KPC-3;

America
States

120
Latin
Brazil
900687
GU: Urine
Surgery
TEM-1(b); CTX-M-14;

America

General
KPC-2;

121
Latin
Brazil
900765
GU: Urine
Emergency
SHV-11(b); TEM-1(b);

America

Room
CTX-M-14; KPC-2;

122
Europe
Greece
921177
GU: Ureter
Medicine
SHV-12(e); TEM-OSBL(u);

General
CMY-13; KPC-2; VIM-1;

123
Europe
Greece
927901
GU: Urine
Medicine
SHV-12(e);

General

124
Europe
Greece
927949
GU: Urine
Medicine
SHV-12(e); TEM-OSBL(u);

General
KPC-2;

125
Europe
Greece
927981
GU: Urine
Medicine
SHV-12(e); TEM-OSBL(u);

General
KPC-2;

126
Europe
Greece
928020
GU: Urine
Pediatric
SHV-12(e); TEM-OSBL(u);

General
KPC-2;

127
Europe
Russia
976026
GU: Urine
Surgery ICU
SHV-OSBL(u); TEM-

OSBL(u); CTX-M-15;

OXA-244(c)

128
North
United
978959
GU: Urine
Medicine
SHV-OSBL(u); TEM-

America
States

General
OSBL(u); KPC-3;

129
Europe
Belgium
979620
GU: Urine
Medicine
SHV-OSBL(u); NDM-1;

General
OXA-232(c)

130
Latin
Brazil
990975
GU: Urine
Medicine
SHV-OSBL(u); TEM-

America

General
OSBL(u); CTX-M-15;

KPC-2;

131
Latin
Brazil
991020
GU: Urine
Medicine ICU
SHV-OSBL(u); TEM-

America

OSBL(u); KPC-2;

132
Latin
Brazil
991969
GU: Urine
Medicine ICU
SHV-OSBL(u); KPC-2;

America

133
Latin
Colombia
960227
GU: Urine
Medicine
SHV-OSBL(u); TEM-

America

General
OSBL(u); KPC-3;

134
South
Australia
1035778
GU: Urine
Surgery
SHV-OSBL(u); TEM-

Pacific

General
OSBL(u); IMP-4;

135
Europe
Russia
1048991
GU: Urine
Surgery
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-15;

OXA-48(c)

136
Latin
Colombia
1109206
GU: Urine
Other

America

137
Latin
Mexico
1138246
GU: Urine
Medicine

America

General

138
Africa
Nigeria
1143069
GU: Urine
Emergency

Room

139
Africa
Nigeria
1143099
GU: Urine
Medicine

General

140
Middle
Kuwait
1143576
GU: Urine
Emergency

East

Room

141
South
Philippines
850792
Bodily Fluids: Abcess/
Other
SHV-1(b); TEM-1(b); CTX-

Pacific

Pus

M-15;

142
South
Philippines
845353
Respiratory: Sputum
Other
SHV-1(b); CTX-M-15;

Pacific

143
South
Philippines
845587
Respiratory: Sputum
Medicine
SHV-31(e);

Pacific

General

144
Europe
Italy
848597
Respiratory: Sputum
Surgery ICU
SHV-12(e);

145
Europe
Czech
851661
Respiratory: Sputum
Medicine ICU
SHV-5(e);

Republic

146
North
United
851698
GI: Stomach
Surgery
SHV-11(b); DHA-1;

America
States

General

147
North
United
851702
GI: Small Colon
Surgery
SHV-12(e); TEM-1(b);

America
States

General

148
Latin
Mexico
854739
Respiratory: Other
Pediatric
SHV-11(b); TEM-1(b);

America

General
CTX-M-15; DHA-1;

149
South
Philippines
855930
Respiratory:
Surgery
SHV-11(b); TEM-1(b);

Pacific

Endotracheal aspirate
General
CTX-M-15;

150
Latin
Venezuela
858492
Respiratory: Sputum
Emergency
SHV-5(e); CTX-M-3;

America

Room

151
Latin
Venezuela
866356
Bodily Fluids:
None Given
SHV-5(e); CTX-M-15;

America

Peritoneal

152
Middle
Israel
869315
GU: Urethra
Medicine
SHV-11(b); TEM-1(b);

East

General
CTX-M-15;

153
Europe
Russia
870216
Respiratory: Sputum
Medicine
SHV-11(b); TEM-1(b);

General
CTX-M-14;

154
Europe
Russia
870245
Respiratory: Sputum
Medicine
SHV-11(b); TEM-1(b);

General
CTX-M-15;

155
Europe
Russia
870258
INT: Wound
Surgery
SHV-11(b); CTX-M-55;

General

156
Asia
China
871354
Respiratory: Sputum
Other
SHV-11(b); TEM-1(b);

CTX-M-15;

157
North
United
873464
Respiratory:
Medicine
SHV-1(b); TEM-1(b); CTX-

America
States

Endotracheal aspirate
General
M-15;

158
Europe
Russia
874317
Respiratory: Sputum
General
SHV-11(b); TEM-1(b);

Unspecified
CTX-M-28;

ICU

159
Europe
Russia
874882
Respiratory:
Medicine ICU
SHV-1(b); TEM-1(b); CTX-

Bronchoalveolar

M-15;

lavage

160
Europe
Russia
874899
Respiratory:
Surgery
SHV-11(b); CTX-M-15;

Bronchoalveolar
General

lavage

161
Europe
Russia
874907
Respiratory:
Medicine
SHV-11(b); TEM-1(b);

Bronchoalveolar
General
CTX-M-15; CMY-2;

lavage

162
Europe
Russia
874921
Respiratory:
Medicine ICU
SHV-12(e);

Bronchoalveolar

lavage

163
Africa
South
884275
Respiratory:
Pediatric ICU
SHV-11(b); TEM-1(b);

Africa

Endotracheal aspirate

CTX-M-15; CTX-M-2;

164
Africa
South
884335
Respiratory: Sputum
Medicine
SHV-OSBL(u); TEM-

Africa

General
OSBL(u); CTX-M-15;

165
Latin
Brazil
900685
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-12(e);

America

Endotracheal aspirate

CTX-M-15;

166
Europe
Greece
921041
INT: Wound
Medicine
SHV-12(e);

General

167
Europe
France
921564
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

Endotracheal aspirate

OSBL(u); CTX-M-15;

168
North
United
928335
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America
States

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

169
North
United
928336
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America
States

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

170
Europe
Portugal
938166
Respiratory: Sputum
Surgery
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-15;

OXA-48(c)

171
Europe
Portugal
938170
Respiratory:
Surgery ICU
SHV-12(e); TEM-OSBL(u);

Endotracheal aspirate

CTX-M-14;

172
Europe
Romania
938937
INT: Wound
Medicine
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-15;

173
Asia
Taiwan
949399
Respiratory: Other
Medicine ICU
SHV-12(e);

174
Latin
Mexico
950099
Respiratory: Sputum
Medicine
SHV-12(e);

America

General

175
Latin
Mexico
950104
Respiratory:
Surgery
SHV-12(e);

America

Endotracheal aspirate
General

176
South
Philippines
957823
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Pacific

Endotracheal aspirate

OSBL(u); CTX-M-15;

177
South
Philippines
957905
Respiratory:
Medicine
SHV-12(e); TEM-OSBL(u);

Pacific

Endotracheal aspirate
General
CTX-M-15; DHA-1;

178
South
Philippines
957920
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Pacific

Endotracheal aspirate

OSBL(u); CTX-M-15;

179
South
Philippines
957921
Respiratory: Sputum
Medicine
SHV-OSBL(u); TEM-

Pacific

General
OSBL(u); CTX-M-15;

180
Asia
Taiwan
958118
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

181
Europe
Russia
975237
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

182
Europe
Russia
975238
Respiratory:
Pediatric ICU
SHV-5(e); CTX-M-15;

Bronchoalveolar

lavage

183
Europe
Russia
975905
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

Bronchoalveolar

OSBL(u); CTX-M-15;

lavage

DHA-1;

184
Europe
Russia
975906
Respiratory:
Surgery ICU
SHV-12(e);

Bronchoalveolar

lavage

185
Europe
Russia
977430
GU: Urine
Surgery
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-15;

186
Europe
Poland
985195
Respiratory: Sputum
Medicine
SHV-12(e); TEM-OSBL(u);

General

187
Asia
Korea,
990759
Respiratory: Sputum
Medicine
SHV-OSBL(u); TEM-

South

General
OSBL(u); CTX-M-15;

188
Asia
Korea,
990778
Respiratory: Sputum
Medicine ICU
SHV-OSBL(u);

South

189
Asia
Malaysia
993593
GU: Urine
Pediatric
SHV-OSBL(u); TEM-

General
OSBL(u); CTX-M-15;

190
Middle
Israel
1007663
Respiratory:
Medicine
SHV-OSBL(u); TEM-

East

Endotracheal aspirate
General
OSBL(u); CTX-M-15;

191
Middle
Israel
1007677
Bodily Fluids:
Surgery
SHV-OSBL(u); TEM-

East

Peritoneal
General
OSBL(u); CTX-M-15;

192
Europe
Russia
1049171
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

Endotracheal aspirate

OSBL(u); CTX-M-15;

193
Europe
Russia
1049223
Respiratory:
Surgery
SHV-OSBL(u); CTX-M-15;

Bronchoalveolar
General

lavage

194
Europe
Russia
1049540
Respiratory: Sputum
Medicine

General

195
Europe
Russia
1049545
Respiratory:
Surgery

Bronchoalveolar
General

lavage

196
Europe
Russia
1049870
Respiratory:
Medicine
SHV-2A(e); TEM-1(b);

Bronchoalveolar
General
CTX-M-15;

lavage

197
Latin
Venezuela
1073801
Respiratory:
Medicine ICU
SHV-36(u);

America

Endotracheal aspirate

198
Asia
Korea,
1085618
Respiratory: Sputum
Medicine
CTX-M-15;

South

General

199
North
United
873460
Respiratory: Sputum
Medicine
SHV-11(b); CTX-M-15;

America
States

General

200
Europe
Russia
874383
Respiratory:
General
SHV-28(e); TEM-1(b);

Endotracheal aspirate
Unspecified
CTX-M-15; DHA-1;

ICU

201
Europe
Russia
875080
Respiratory: Sputum
Surgery ICU
SHV-12(e); TEM-OSBL(u);

202
North
United
882753
INT: Wound
Medicine
SHV-OSBL(u); TEM-

America
States

General
OSBL(u); CTX-M-15;

203
Asia
Taiwan
894334
Respiratory: Other
Medicine ICU
SHV-12(e);

204
Europe
Czech
923859
Respiratory:
Surgery ICU
SHV-12(e);

Republic

Endotracheal aspirate

205
Europe
Portugal
938167
Bodily Fluids:
Emergency
SHV-OSBL(u); TEM-

Peritoneal
Room
OSBL(u); CTX-M-15;

206
Latin
Mexico
950100
Respiratory:
Medicine ICU
SHV-OSBL(u); CTX-M-14;

America

Bronchoalveolar

lavage

207
Latin
Mexico
950102
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

America

Endotracheal aspirate

OSBL(u); CTX-M-15;

208
Latin
Mexico
950107
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America

Endotracheal aspirate

OSBL(u); CTX-M-15;

209
Latin
Mexico
951241
Respiratory:
Pediatric ICU
SHV-OSBL(u); TEM-

America

Bronchial brushing

OSBL(u); CTX-M-15;

210
South
Philippines
957918
Respiratory: Sputum
Medicine ICU

Pacific

211
South
Philippines
966430
Respiratory: Sputum
Medicine

Pacific

General

212
Europe
Russia
975841
Respiratory: Sputum
Surgery ICU
SHV-OSBL(u); TEM-

OSBL(u); CTX-M-15;

213
Latin
Mexico
979791
Respiratory:
Surgery ICU
SHV-OSBL(u); TEM-

America

Bronchial brushing

OSBL(u); CTX-M-15;

214
North
United
981436
Respiratory:
Medicine ICU
SHV-OSBL(u); TEM-

America
States

Endotracheal aspirate

OSBL(u); CTX-M-15;

215
Europe
Russia
1049468
Respiratory:
Medicine
SHV-110(u); CTX-M-15;

Bronchoalveolar
General

lavage

216
Europe
Russia
1050019
INT: Wound
Surgery ICU
SHV-OSBL(u); TEM-

OSBL(u); CTX-M-15;

217
Europe
Russia
1050029
INT: Wound
General

Unspecified

ICU

218
Europe
Russia
1050038
INT: Wound
Medicine

General

219
Europe
Russia
1050052
Respiratory:
Medicine ICU

Bronchoalveolar

lavage

220
North
United
1073361
Respiratory: Sputum
Medicine
TEM-1(b); CTX-M-15;

America
States

General

221
Europe
Spain
1073953
Respiratory: Sputum
Medicine
SHV-11(b); CTX-M-15;

General

222
Europe
United
1081745
Respiratory:
Medicine ICU
SHV-11(b); TEM-1(b);

Kingdom

Endotracheal aspirate

CTX-M-15;

223
Latin
Mexico
950095
Respiratory:
Surgery ICU
SHV-12(e); DHA-1;

America

Endotracheal aspirate

224
Europe
Russia
870321
Respiratory: Other
Surgery
SHV-OSBL(u);

General

225
Europe
Russia
1050017
INT: Wound
Surgery ICU

226
Europe
Russia
1050053
Respiratory:
Medicine ICU

Bronchoalveolar

lavage

227
Europe
Greece
848879
Respiratory: Sputum
General

Unspecified

ICU

228
North
United
855499
INT: Wound
Outpatient

America
States

229
Latin
Colombia
860723
GI: Abscess
General

America

Unspecified

ICU

230
Asia
Taiwan
862260
Respiratory: Sputum
Surgery

General

231
North
United
863922
GI: Gall Bladder
Other

America
States

232
Latin
Venezuela
866365
GU: Urine
Medicine ICU

America

233
Europe
Austria
896420
INT: Wound
Surgery ICU

234
North
United
920265
GI: Stomach
Medicine ICU

America
States

235
Asia
Thailand
949027
Respiratory: Sputum
Medicine

General

236
Latin
Brazil
991831
GU: Urine
Emergency

America

Room

237
Asia
Malaysia
996710
GI: Abscess
Surgery

General

238
North
United
1072091
GU: Urine
Medicine ICU

America
States

239
Europe
United
1087502
Respiratory: Sputum
Surgery

Kingdom

General

240
Africa
South
1088186
INT: Wound
Medicine ICU

Africa

241
Europe
Belgium
1089861
GU: Urine
Medicine

General

242
Europe
Czech
1097531
Respiratory:
Medicine ICU

Republic

Endotracheal aspirate

243
Europe
Spain
1098542
INT: Wound
Medicine

General

244
Asia
Japan
1132015
Respiratory: Sputum
Medicine

General

245
Europe
France
1149311
Bodily Fluids:
Medicine ICU

Peritoneal

246
Asia
Hong
1151042
GU: Urine
Medicine

Kong

General

247
South
Philippines
845578
INT: Wound
Medicine

Pacific

General

248
Europe
Portugal
845918
INT: Wound
Medicine

General

249
Latin
Chile
847189
GI: Abscess
Surgery ICU

America

250
Latin
Argentina
847386
GI: Abscess
Surgery

America

General

251
North
United
857611
Respiratory: Sputum
Surgery ICU

America
States

252
Latin
Colombia
860724
Respiratory: Sputum
Medicine

America

General

253
Latin
Venezuela
866345
Respiratory: Sputum
Medicine

America

General

254
Latin
Argentina
867249
Respiratory:
Surgery ICU

America

Endotracheal aspirate

The isolates in rows 1-140 of Table 8 are Klebsiella pneumoniae carbapenamase (KPC) strains. The isolates in rows 141-226 of Table 8 are extended spectrum beta lactamase (ESBL) strains, and the isolates in rows 227-254 of Table 8 are antibiotic-susceptible strains.

These results demonstrate that anti-O2 mAbs bind not only to a large and diverse group of clinical strains, but also to antibiotic resistant clinically relevant strains. These results suggest that anti-O2 mAbs can be useful as a therapeutic and/or diagnostic as described herein, for example, for one or more of the Klebsiella strains disclosed in Table 8.

Example 14: Anti-O2 Antigen Antibodies Bind to O2 Klebsiella Strains Regardless of gml Gene Expression

Monoclonal antibodies that recognized Gal III structure have been reported to bind to gml+Klebsiella ST258 strains. We obtained whole genome sequences from a number of O2 Klebsiella clinical isolates and performed experiments to (i) whether these strains express the gml gene; and (ii) whether anti-O2 mAbs bind to these strains by fluorescence activated cell sorting (FACS) or western blot analysis. In addition, we analyzed multi locus sequence type (MLST) and gml locus based on whole genome sequences. Table 9 lists FACS binding, MLST, and gml expression data of 31 O2 Klebsiella clinical isolates.

TABLE 9

Anti-O2 monoclonal antibodies binding to gml+

and gml− Klebsiella clinical isolates.

KPN42
KPN70
KPN179
MLST
gml
comments

ARC2698
+
+
+
258
+

ARC2708
+
+
+
258
+

ARC2712
+
+
+
258
+

ARC2929
+
+
+
258
+

ARC2945
+
+
+
258
+

ARC3516
+
+
+
45

ARC3797
+
+
+
147

ARC4771
+
+
+
45

ARC4773
+/−
+/−
+/−
45

KPN42+ by

Western

ARC4778
+
+
+
70
+

ARC4780
+
+
+
11
+

ARC4784
+
+
+
218
+

ARC5109
+
+
+
258
+

ARC5113
+
+
+
1728

KPN42+ by

Western

ARC5115
+
+
+
34

ARC5116
+
+
+
258
+

ARC5117
+
+
+
258
+

ARC5121
+
++
+
258
+

ARC5372
+
+
+
258
+
KPN42+ by

Western

ARC5411
+
+
+
258
+

ARC5449
+
+
+
11*

KPN42+ by

Western

ARC6084
+
+
+
17
+
KPN42+ by

Western

ARC6086
+
+
+
11
+

ARC6093
+
+
+
11

ARC6095
+
+
+
258
+
KPN42+ by

Western

ARC6099
+
+
+
437*

ARC6100
+
+
+
340

ARC6102
+
+
+
20
+
KPN42+ by

Western

ARC6106
+
+
+
641

ARC6114
+
+
+
45

ARC6118
+
+
+
512
+

As shown in Table 9, multiple ST types, including ST258, were present in this collection. Twelve of the non-ST258 strains did not express the gml gene. KPN42 bound to 92% (11/12) gml− and 100% (19/19) gml+ Klebsiella strains. These data suggest that anti-O2 antibodies such as KPN42 bind to O2 antigen, but do not bind to a Gal III epitope. Thus, such O2 antibodies confer broader coverage against O2 strains than Gal III-binding antibodies.

Example 15: KPS44 Sequence Optimization

In order to reduce sequence liability for mAb development, a tryptophan in the heavy chain CDR3 of KPS44 was exchanged with a phenylalanine (see heavy chain of KPS44-v2017, SEQ ID NO: 202, also known as KPS44-v2017-W108F-VH). Additionally, a tryptophan in the light chain CDR1 of KPS44 was exchanged with a phenylalanine, and an aspartic acid and a serine in the light chain CDR3 of KPS44 were substituted for an asparagine and a tyrosine, respectively (see light chain of KPS44v-2017, SEQ ID NO: 203, also known as KPS44-v2017-D37N-S38Y-W107F-VL). Collectively, this resulted in KPS44-v2017 (also known as KPS44-W108F-VH/KPS44-D37N-S38Y-W107F-VL.)

Combinations of the heavy chain CDRs and light chain CDRs of parental KPS44 and KPS44-v2017 were made, resulting in KPS44-D37N-S38Y-W107F-VL (KPS44-G1), KPS44-W108F-VH/KPS44-W107F-VL (KPS44-G2), and KPS44-W108F-VH/KPS44-D37N-S38Y-VL (KPS44-G3).

The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

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Anti-O2 antibodies and uses thereof

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