Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
  • A61K39/025—Enterobacteriales, e.g. Enterobacter
  • A61K39/0266—Klebsiella
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/40—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum bacterial
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

• MRKA-100-WO-PCT_SeqListing.txt (Size: 42, 157 bytes; and Date of Creation: August 16, 2016) filed with the application is incorporated herein by reference in its entirety.
• the field of the invention generally relates to MrkA polypeptides, MrkA-encoding polynucleotides, and anti-MrkA antibodies for prevention or treatment of Klebsiella infections.
• Klebsiella is a Gram negative bacterium that is rapidly gaining clinical importance as a causative agent for optimistic and nosocomial infection, including pneumonia, urinary tract infection, neonatal septicemia, and surgery wound infection.
• Klebsiella infections such as pyogenic liver abscesses (PL A), endophthalmitis, meningitis, and necrotizing meningitis.
• opportunistic infections are most problematic and call for novel antimicrobial drugs ⁇ see, e.g., Xu et al., Expert opinion on investigational drugs 2014; 23 : 163-82).
• Klebsiella pneumoniae a causative agent for opportunistic and nosocomial infections (Broberg et al., FlOOOPrime Rep 2014; 6:64), has become particularly challenging with multi-drug resistant strains widely circulating.
• Klebsiella infections 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.
• K. pneumoniae including genomics and proteomics approaches (Lundberg et al., Hum Vaccin Immunother 2012; 9:497-505; Meinke et al., Vaccine 2005; 23 :2035-41; Maroncle et al., Infection and immunity 2002; 70:4729-34).
• targets have been suggested from these studies, few have been validated through subsequent investigations.
• the majority of potential targets identified through such approaches are proteins involved in metabolic pathways which may not be suitable as antibody targets due to inaccessibility.
• Antigenome strategy represents a novel approach to identify directly antigens capable of eliciting antibody responses (Meinke et al. 2005, Vaccine, 23(17-18):2035-41). Its impact on K. pneumoniae investigation remains to be seen.
• type III fimbriae mediate biofilm formation on biotic and abiotic surfaces and are required for mature biofilm development.
• the various components of type 3 fimbriae are encoded by the mrkABCDF operon, which produce the major pilin sub unit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF. See Yang et al. PLoS One. 2013 Nov 14;8(1 l):e79038.
• MrkA binding proteins e.g., antibodies or antigen binding fragments thereof, that bind to and induce opsonophagocytic killing (OPK) of Klebsiella.
• the present disclosure also provides methods of treating Klebsiella infections using MrkA binding proteins, e.g., antibodies or antigen binding fragments thereof, MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof.
• MrkA binding proteins e.g., antibodies or antigen binding fragments thereof, MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof.
• MrkA binding proteins e.g., antibodies or antigen binding fragments thereof, MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof.
• an isolated antigen binding protein that specifically binds to MrkA, wherein the antigen binding protein a) binds to at least two Klebsiella pneumoniae (K. pneumoniae) serotypes; b) induce
• the antigen binding protein binds to at least two K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the antigen binding protein induces OPK in at least one or two K.
• the antigen binding protein induces 100% OPK iri K. pneumoniae strains 9148 (02a:K28), 9178 (03 :K58), and 9135 (04:K15); and/or induces 80% OPK in K. pneumoniae strain 29011 (01 :K2) as measured using a bio- luminescent OPK assay.
• the antigen binding protein confers survival benefit in an animal exposed to a K. pneumoniae strain selected from the group consisting of Kp29011, Kp9178, and Kp43816.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA, wherein the antigen binding protein inhibits biofilm formation.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA, wherein the antigen binding protein inhibits cell attachment.
• an isolated antigen binding protein that has
• HCDRl has the amino acid sequence of SEQ. ID. NO: 1
• HCDR2 has the amino acid sequence of SEQ. ID. NO: 2
• HCDR3 has the amino acid sequence of SEQ. ID. NO: 3
• LCDRl has the amino acid sequence of SEQ. ID. NO: 7
• LCDR2 has the amino acid sequence of SEQ. ID. NO: 8
• LCDR3 has the amino acid sequence of SEQ. ID. NO: 9.
• an isolated antigen binding protein that has
• the antigen binding protein comprises a heavy chain variable region (VH) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13 and/or a light chain variable region (VL) at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 15.
• the antigen binding protein thereof comprises a VH comprising SEQ ID NO: 13 and a VL comprising SEQ ID NO: 15.
• an isolated antigen binding protein that specifically binds to MrkA comprising a VH comprising SEQ ID NO: 13.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA comprising a VL comprising SEQ ID NO: 15.
• an isolated antigen binding protein that has
• HCDR1 has the amino acid sequence of SEQ. ID. NO: 4
• HCDR2 has the amino acid sequence of SEQ. ID. NO: 5
• HCDR3 has the amino acid sequence of SEQ. ID. NO: 6
• LCDR1 has the amino acid sequence of SEQ. ID. NO: 10
• LCDR2 has the amino acid sequence of SEQ. ID. NO: 11
• LCDR3 has the amino acid sequence of SEQ. ID. NO: 12.
• an isolated antigen binding protein that has
• the antigen binding protein comprises a heavy chain variable region (VH) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14 and/or a light chain variable region (VL) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16.
• VH heavy chain variable region
• VL light chain variable region
• the antigen binding protein comprises a VH comprising SEQ ID NO: 14 and a VL comprising SEQ ID NO: 16.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA comprising a VH comprising SEQ ID NO: 14.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA comprising a VL comprising SEQ ID NO: 16.
• the antigen binding protein binds to an epitope in amino acids 1-
• the antigen binding protein specifically binds to MrkA (SEQ ID NO: 17), but does not bind to either SEQ ID NO:26 (MrkA lacking amino acids 1- 40 of SEQ ID NO: 17) or SEQ ID NO:27 (MrkA lacking amino acids 171-202 of SEQ ID NO: 17).
• an isolated antigen binding protein that has
• an isolated antigen binding protein that has
• an isolated antigen binding protein that specifically binds to MrkA comprising a set of Complementarity-Determining Regions (CDRs): HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 selected from the group consisting of: (i) SEQ ID NOs: 29-31 and 41-43, respectively; (ii) SEQ ID NOs: 32-34 and 44-46, respectively; (iii) SEQ ID NOs: 35-37 and 47-49, respectively; and (iv) SEQ ID NOs: 38-40 and 50-52, respectively.
• CDRs Complementarity-Determining Regions
• an isolated antigen binding protein that has
• said antigen binding protein comprises a heavy chain variable region (VH) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:53, 54, 55, or 56 and/or a light chain variable region (VL) at least 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:57, 58, 59, or 60.
• VH heavy chain variable region
• VL light chain variable region
• the antigen binding protein comprises a VH comprising SEQ ID NO:53, 54, 55, or 56 and a VL comprising SEQ ID NO:57, 58, 59, or 60.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA comprising a VH comprising SEQ ID NO:53, 54, 55, or 56.
• an isolated antigen binding protein that has
• MrkA specifically binds to MrkA comprising a VL comprising SEQ ID NO:57, 58, 59, or 60.
• an isolated antigen binding protein that specifically binds to the same MrkA epitope as an antibody or antigen-binding fragment thereof selected from the group consisting of: (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO: 13 and a light chain variable region (VL) comprising SEQ ID NO: 15; (b) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 16; (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO:53 and light chain variable region (VL) comprising SEQ ID NO:57; (d) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO:54 and light chain variable region (VL) comprising SEQ ID NO:58;
• an isolated antigen binding protein that competitively inhibits binding of a reference antibody to MrkA, wherein said reference antibody is selected from the group consisting of: (a) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO: 13 and a light chain variable region (VL) comprising SEQ ID NO: 15; (b) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO: 14 and a light chain variable region (VL) comprising SEQ ID NO: 16; (c) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO:53 and light chain variable region (VL) comprising SEQ ID NO:57; (d) an antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising SEQ ID NO:54 and light chain variable region (VL) comprising SEQ ID NO:58; (e)
• the antigen binding protein or antigen-binding fragment thereof binds oligomeric MrkA.
• an isolated antigen binding protein that has
• the antigen binding protein is murine, non-human, humanized, chimeric, resurfaced, or human.
• the antigen binding protein is an antibody.
• the antigen binding protein is a monoclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a bi-specific antibody, a multi-specific antibody, or an antigen binding fragment thereof.
• the antigen binding protein is an antigen binding fragment of an antibody.
• the antigen binding protein comprises a Fab, Fab', F(ab')2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody,
• the antigen binding protein comprises a Fab, Fab', F(ab')2, single chain Fv or scFv, disulfide linked Fv, intrabody, IgGACKZ, minibody, F(ab')3, tetrabody, triabody, diabody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.
• the antigen binding protein binds to MrkA with a Kd of about 1.0 nM to about 10 nM. In one instance, the antigen binding protein binds to MrkA with a Kd of 1.0 nM or less. In one instance, the binding affinity is measured by flow cytometry, Biacore, KinExa, radioimmunoassay, or bio-layer interferometry (BLI).
• the antigen binding protein a binds to at least two Klebsiella
• K pneumoniae (K pneumoniae) serotypes; b) induces opsonophagocytic killing (OPK) of K. pneumoniae or c) binds to at least two K. pneumoniae serotypes and induces OPK of K.
• OPK opsonophagocytic killing
• the antigen binding protein (including, e.g., an anti-MrkA
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces Kp43816 biofilm formation.
• the antigen binding protein (including, e.g., an anti-MrkA
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces Klebsiella cell attachment.
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces Klebsiella (including, e.g., Kp43816) cell attachment to a human cell.
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces or reduces
• Klebsiella including, e.g., Kp43816) cell attachment to human epithelial cells.
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces Klebsiella (including, e.g., Kp43816) cell attachment to pulmonary epithelial cells.
• the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) inhibits or reduces Klebsiella (including, e.g., Kp43816) cell attachment to pulmonary epithelial cells.
• the antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• Klebsiella including, e.g., Kp43816, cell attachment to A549 cells.
• the antigen binding protein comprises a heavy chain
• the antigen binding protein comprises 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 comprises a human IgGl constant domain and a human lambda constant domain.
• the antigen binding protein comprises an IgGl constant domain.
• the antigen binding protein comprises an IgGl/IgG3 chimeric constant domain.
• a hybridoma producing the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof).
• an isolated host cell producing the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof).
• an antigen binding protein comprising (a) culturing a host cell expressing said antigen binding protein; and (b) isolating said antigen binding protein thereof from said cultured host cell.
• an antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• the present disclosure also provides a pharmaceutical composition
• a pharmaceutical composition comprising the antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) and a pharmaceutically acceptable excipient.
• the pharmaceutically acceptable excipient is a preservative, stabilizer, or antioxidant.
• the pharmaceutical composition is for use as a medicament.
• the antigen binding protein or the pharmaceutical composition further comprises a labeling group or an effector group.
• 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), and yellow fluorescent proteins (YFPs), and polypeptide epitopes recognized by a secondary reporter such as histidine peptide (his), hemagglutinin (HA), gold binding peptide, and Flag.
• the effector group is selected from the group consisting of a radioisotope, radionuclide, a toxin, a therapeutic and a chemotherapeutic agent.
• an antigen binding protein [0043] In one instance, provided herein is the use of an antigen binding protein
• composition for treating or preventing a condition associated with a Klebsiella infection.
• the present disclosure also provides a method for treating, preventing, or
• an antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• an antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• Klebsiella in a subject comprising administering to a subject in need thereof an antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) or pharmaceutical composition provided herein.
• an antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• pharmaceutical composition provided herein.
• provided herein is a method for treating, preventing, or
• the condition is selected from the group consisting of pneumonia, urinary tract infection, septicemia, neonatal septicemia, 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.
• the condition is a nosocomial infection.
• the Klebsiella is K pneumonia, K oxytoca, K planticola and/or K granulomatis. In one instance, the Klebsiella is resistant to cephalosporin, aminoglycoside, quinolone, and/or carbapenem. In one instance, the method further comprises administering an antibiotic. In one instance, the antibiotic is a carbapanem or colistin.
• Klebsiella in a subject comprising administering to a subject in need thereof an effective amount of an anti-MrkA antibody or an antigen binding fragment thereof.
• the anti-MrkA antibody or antigen binding fragment thereof specifically binds to K pneumonia, K oxytoca, K planticola and/or K granulomatis MrkA.
• the anti-MrkA antibody or antigen binding fragment thereof specifically bins to K
• the present disclosure also provides an isolated nucleic acid molecule encoding an antigen binding protein provided herein.
• an isolated nucleic acid molecule encoding a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 13, 14, 53, 54, 55, and 56.
• an isolated nucleic acid molecule encoding a light chain variable region (VL) sequence selected from the group consisting of SEQ ID NOs: 15, 16, 57, 58, 59, and 60.
• the nucleic acid molecule is operably linked to a control
• provided herein is a vector comprising a nucleic acid molecule provided herein.
• a host cell transformed with a nucleic acid molecule provided herein or a vector provided herein.
• a host cell transformed with a nucleic acid encoding a heavy chain variable region (VH) sequence selected from the group consisting of SEQ ID NOs: 13, 14, 53, 54, 55, and 56 and a nucleic acid molecule encoding a VL sequence selected from the group consisting of SEQ ID NOs: 15, 16, 57, 58, 59, and 60.
• VH heavy chain variable region
• the host cell is a mammalian host cell.
• the host cell is a NS0 murine myeloma cell, a PER.C6 ® human cell, or a Chinese hamster ovary (CHO) cells.
• the present disclosure also provides a pharmaceutical composition
• a pharmaceutical composition comprising
• the disclosure provides a vaccine comprising MrkA, an immunogenic fragment thereof, or a polynucleotide encoding MrkA or an immunogenic fragment thereof.
• the pharmaceutical composition or vaccine comprises an immunologically effective amount of the MrkA, immunogenic fragment thereof, or polynucleotide encoding MrkA or an immunogenic fragment thereof.
• the pharmaceutical composition or vaccine comprises an adjuvant.
• the MrkA or immunogenic fragment thereof of the pharmaceutical composition or vaccine is monomeric.
• the MrkA or immunogenic fragment thereof of the pharmaceutical composition or vaccine is oligomeric.
• the MrkA is K.
• the MrkA or immunogenic fragment thereof comprises a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17 or wherein the polynucleotide encoding MrkA or an immunogenic fragment thereof encodes a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17.
• the MrkA or immunogenic fragment thereof comprises the sequence set forth in SEQ ID NO: 17 or wherein the polynucleotide encoding MrkA or an immunogenic fragment thereof encodes the sequence set forth in SEQ ID NO: 17.
• the present disclosure also provides a method of inducing an immune response against Klebsiella in a subject comprising administering to the subject a pharmaceutical composition, a MrkA or immunogenic fragment thereof, or vaccine provided herein.
• the immune response comprises an antibody response.
• the immune response comprises a cell-mediated immune response.
• the immune response comprises a cell-mediated immune response and an antibody response.
• the immune response is a mucosal immune response.
• the immune response is a protective immune response.
• Klebsiella comprising administering to a subject the pharmaceutical composition, MrkA or immunogenic fragment thereof, or vaccine provided herein.
• a method for treating, preventing, or reducing the incidence of a condition associated with a Klebsiella infection in a subject in need thereof comprising administering to said subject MrkA, an immunogenic fragment thereof, or a polynucleotide encoding MrkA or an immunogenic fragment thereof.
• a method for inhibiting the growth of Klebsiella in a subject comprising administering to a subject in need thereof MrkA, an immunogenic fragment thereof, or a polynucleotide encoding MrkA or an immunogenic fragment thereof.
• the methods provided herein the methods provided herein, the
• Klebsiella is K pneumonia, K oxytoca, K. planticola and/or K granulomatis. In one instance, the Klebsiella is K pneumonia. In one instance of the methods provided herein, the MrkA or immunogenic fragment thereof is monomelic. In one instance of the methods provided herein, the MrkA or immunogenic fragment thereof is oligomeric. In one instance of the methods provided herein, the MrkA is K pneumonia MrkA. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
• FIGS 1A-F depict the K. pneumoniae binding and potent OPK activity of
• mAbs monoclonal antibodies isolated through phage and hybndoma platforms.
• Antibody binding to Kp29011 in a whole cell ELISA assay two hybndoma clones (88D10 and 89E10) and two phage antibodies (Kp3 and Kpl6) bind to K. pneumoniae strain 29011 in ELISA assays as described in Example 2. As expected, control antibody hlgG control did not bind to K. pneumoniae strain 29011.
• Phage antibodies (Kp3 and Kpl6) compete for binding to K. pneumoniae. One ⁇ g/ml of biotin-labeled Kp3 was mixed with increasing amount of unlabeled phage and control antibodies as indicated and tested for its binding to K. pneumoniae strain 29011.
• Streptavidin-HRP was used as the detecting agent.
• Kp3 and Kpl6 both prevented binding of biotin-labeled Kp3 to K. pneumoniae strain 29011.
• D Phage (Kp3 and Kpl6) and hybridoma antibodies (88D10) compete in binding to K. pneumoniae.
• One ⁇ g/ml of hybridoma clone 88D10 was mixed with increasing amount of phage and control antibodies (hlgG) and tested for its binding to K. pneumoniae strain 29011.
• Anti-mouse-IgG-HRP was used as the detecting agent. The reduction in ELISA signal was expressed as a percentage of inhibition.
• Kp3 and Kpl6 both prevented binding of 88D10 to K. pneumoniae strain 2901.
• E Phage (Kp3 and Kpl6) and hybridoma antibodies (88D10) compete in binding to K. pneumoniae.
• One ⁇ g/ml of hybridoma clone 88D10
• Phage (Kp3 and Kpl6) and hybridoma (21G10, 22B12, 88D10 and 89E10) antibodies bind to K. pneumoniae strains with various serotypes. "+” indicates binding.
• F. Anti-MrkA mAb Kp3 displays potent OPK activity against K. pneumoniae of different serotypes.
• Figures 2A-D depict the results of experiments identifying MrkA as the antigen bound by K. pneumoniae specific antibodies generated herein.
• Lane 1 pre-stained molecular weight marker
• Lane 2 cell lysate from Kp3 nonreactive strain 1899
• Lane 3 cell lysate from Kp3 reactive strain 43816DM
• Lane 4 - 1899 lysate subjected to immunoprecipitation by isotype control
• Lane 5 - 1899 lysate subjected to immunoprecipitation by Kp3
• Lane 6 - 1899 lysate subjected to
• A Recombinant expression of MrkA by Western blot analysis using anti-his tag (left panel) and Kp3 (right panel) antibodies. Lane 1 : host cell only; Lane 2: host cell transformed with empty vector; Lane 3 : host cell transformed with expression vector carrying his-tagged MrkA ORF; and Lane 4: lysate prepared from strain 43816DM. These results show that Kp3 binds to recombinant MrkA.
• B In vitro transcription and translation of MrkA and Western blot analysis using Kp3 (left panel) anti-Myc tag (right panel) antibodies.
• Figures 4A-D depict the protective activity of Kp3 mAb in various in vivo
• a and B Kp3 reduces organ burden in intranasal lung infection model against Kp29011 (01 :K2) and Kp9178 (03 :K38), respectively.
• An irrelevant human IgGl antibody (hlgGl) and rabbit polyclonal antibody against Kp43816 (Rab IgG) were used as controls. All antibodies were used at a dose of 15 mg/kg. These results show that anti-MrkA antibody Kp3 reduced organ burden when administered prior to bacterial challenge.
• C Kp3 enhanced survival in a lethal bacterial pneumonia model using Kp43816 (01 :K2).
• An irrelevant human IgGl (hlgGl) antibody was used as a control. Both antibodies were used at a dose of 15 mg/kg.
• Figure 5 depicts MrkA conservation among the enterobactereaceae family
• MrkA is conserved among the majority of enter obactereace family members.
• FIG. 6 depicts the results of MrkA binding assays. Full length MrkA ("MrkA-
• MrkA-N-dlt a 40 amino acid N-terminal deletion
• MrkA with a 40 amino acid N-terminal deletion i.e., amino acids 41-202 of SEQ ID NO: 17 (i.e., SEQ ID NO:26)
• MrkA with a 32 amino acid C- terminal deletion MrkA with a 32 amino acid C- terminal deletion
• MrkA with both the N and C terminal deletions MrkA with both the N and C terminal deletions
• MrkA-N/C-dlt i.e., amino acids 41-170 of SEQ ID NO: 17 (i.e., SEQ ID NO:28)
• TopiclO cont an empty vector
• Figure 7 depicts purification of monomeric and oligomeric MrkA. Fractions of monomeric and oligomeric MrkA were expressed, purified, and analyzed by SDS-PAGE gel under reducing and non-reducing conditions and visualized with blue stain. M: molecular weight marker. Lanes 1 and 4 contain monomeric MrkA from pool 1. Lanes 2 and 5 contain monomeric MrkA from pool 2. Lanes 3 and 6 contain oligomeric MrkA.
• FIGs 8A-B shows that MrkA vaccination reduces lung burden.
• C57/bl6 mice immunized with monomeric or oligomeric MrkA were challenged with Kp29011 (01 :K2) intra-nasally.
• Kp29011 01 :K2
• the presence of bacteria in lung and liver were analyzed 24 hours post infection.
• Monomeric MrkA significantly reduced bacteria in the lung ( Figure 8A)
• oligomeric MrkA significantly reduced bacteria in both the lung and liver ( Figure 8B).
• (*) indicates Student's t test p value ⁇ .05.
• FIG. 9 shows that Kp3 inhibits Klebsiella biofilm formation.
• Kp43816 was added to Falcon plastic plates in the presence of the anti-MrkA antibody Kp3 (closed triangles), or hlgGl (isotype control antibodies, open triangles, "R347"). The inhibition of biofilm formation was graphed. (**) indicates Student's t test p value ⁇ 0.01 for Kp3 values relative to isotype control.
• FIG 10 shows that Kp3 inhibits Klebsiella binding to epithelial cells.
• Kp43816 was added to A549 cells (2xl0 5 /well) in the presence of the anti-MrkA antibody Kp3 (closed triangles), or hlgGl (open triangles, "R347"). Samples were run in duplicate; graph is representative of 3 separate experiments.
• (*) indicates Student's t test p value ⁇ .05 for Kp3 values relative to isotype control. Where error bars cannot be seen they are smaller than the symbol width.
• Figure 11 shows the phage panning output screening cascade described in
• Example 10 More than 4000 colonies were picked for high throughput screening after phage panning, scFv.Fc conversion, and transformation. Four clones including clones 1, 4, 5, and 6 were selected for further characterization.
• Figure 12 shows a schematic representation of a four-component homogeneous time resolved FRET (HTRF) used for screening for MrkA binders.
• Component A which is Streptavidin-Eu(K) cryptate and serves as the energy donor, is brought into close proximity of component D, which is anti-huFc-alexa fluor 647 and serves as the energy acceptor by the interaction between components B and C.
• component D which is anti-huFc-alexa fluor 647 and serves as the energy acceptor by the interaction between components B and C.
• B is the biotin-labeled MrkA
• C is a scFv-Fc specific for MrkA.
• FIG 13 shows binding assays using anti-MrkA antibodies. MrkA protein was either coated directly onto the ELISA plate (right panel) or captured by streptavidin after biotinylation (left panel). The MrkA protein was recognized differently by anti-MrkA antibodies in these different antigen-presentation formats.
• Figure 14 shows that anti-MrkA antibodies bind preferably to the oligomeric
• MrkA prepared directly from a KP strain (K) as compared to the recombinant MrkA expressed in E. coli (E) in a Western blot analysis.
• Clone 1 is the only antibody capable of detecting the monomeric MrkA from KP (indicated by an arrow).
• Figure 15 shows the result of epitope binding assays. Epitope binning was
• Figure 16 demonstrates that OPK activity is important for in vivo protective
• FIG. 17 shows serotype-independent binding to KP strains by anti-MrkA antibodies. A flow cytometry experiment was used to gauge the binding of four anti-MrkA antibodies against three WT KP strains of different serotypes. R347 is a human IgG isotype control.
• Figure 18 shows serotype-independent OPK activities by anti-MrkA antibodies.
• Figure 19 shows the results of a prophylactic in vivo challenge model. Antibodies were given 24 hours prior to KP challenge.
• Figure 20 shows the results of a therapeutic in vivo challenge model. Antibodies were given one hour after KP challenge.
• Figure 21 shows that individual antibodies are as effective as antibody
• KP3 was combined with either clone 1 or clone 5 in equal amount as indicated and tested in a therapeutic model.
• the present disclosure provides isolated binding proteins, including antibodies or antigen binding fragments thereof, which bind to MrkA.
• Related polynucleotides, vectors, host cells, and pharmaceutical compositions comprising the MrkA binding proteins, including antibodies or antigen binding fragments thereof, are also provided. Also provided are methods of making and using the MrkA 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 by administering the MrkA binding proteins, including antibodies or antigen binding fragments, disclosed herein.
• 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).
• 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).
• antigen binding protein refers to a molecule comprised of one or more polypeptides that recognizes and specifically binds to a target, e.g., MrkA, such as an anti- MrkA antibody or antigen-binding fragment thereof.
• 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.
• an antibody encompasses intact polyclonal antibodies, intact monoclonal antibodies, multispecific antibodies such as bispecific antibodies generated from at least two intact 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 any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g.
• IgGl, IgG2, IgG3, IgG4, IgAl 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.
• 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.
• antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFvs, and single chain antibodies.
• 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 antibody molecules 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.
• binding fragments are (i) the Fab fragment consisting of VL, VH, CL, and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.
• Fv, scFv or diabody 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).
• bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above.
• bispecific antibodies include those of the BiTETM technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain.
• Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti -idiotypic reaction.
• Bispecific diabodies as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E.coli.
• Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against MrkA, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected.
• Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B.
• Immunoglobulin-like domain-based technologies that have created multispecific and/or multivalent molecules include dAbs, TandAbs, nanobodies, BiTEs, SMTPs, D Ls, Affibodies, Fynomers, Kunitz Domains, Albu-dabs, DARTs, DVD-IG, Covx-bodies, peptibodies, scFv-Igs, SVD-Igs, dAb-Igs, Knobs-in-Holes, DuoBodiesTM and triomAbs.
• Bispecific bivalent antibodies, and methods of making them are described, for instance in U.S. Pat. Nos. 5,731, 168; 5,807,706; 5,821,333; and U.S. Patent Appl. Publ. Nos.
• 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
• an antigen binding protein e.g., an antibody or antigen binding fragment thereof
• an antigen binding protein e.g., an antibody or antigen binding fragment thereof
• an antigen binding protein e.g., an antibody or antigen binding fragment thereof
• contains an alteration in an Fc region e.g., amino acid substitution, deletion, or addition or change in oligosaccharide
• 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).
• an effector function e.g., ADCC, CDC, and/or ADCP
• the term "specific” may 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.
• 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
• the dissociation constant is the binding affinity constant and is given by:
• 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 MrkA may, 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
• OPK activity Assays that can be used to demonstrate OPK activity include the bio-luminescent OPK activity used in the Examples or by counting the bacterial colonies on Agar plates. Additional assays are provided, for example, in DiGiandomenico et a/., J. Exp. Med. 209: 1273-87 (2012), which is incorporated herein by reference.
• 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%.
• antigen binding proteins e.g., neutralizing antigen binding proteins or neutralizing antibodies
• competition 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 MrkA protein or a fragment thereof).
• a reference antigen binding protein e.g., a ligand, or a reference antibody
• RIA solid phase direct or indirect radioimmunoassay
• EIA solid phase direct or indirect enzyme immunoassay
• 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.
• 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.
• 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.
• the test antigen binding protein is present in excess.
• Antigen binding proteins identified by competition assay 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.
• a competing antigen binding protein 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.
• epitopes 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.
• an antigen e.g., a target polypeptide that they recognize or specifically bind.
• the portion of MrkA 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.
• polypeptide refers to a molecule composed of
• polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product.
• 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.
• 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.
• 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.
• 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 851 10503) 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.
• 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
• Recombinantly produced 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.
• fragments, variants, or derivatives of polypeptides are also included in the present disclosure.
• 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.
• variant 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.
• 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).
• a second polypeptide or another molecule e.g., a polymer such as PEG, a chromophore, or a fluorophore
• atom e.g., a radioisotope
• 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).
• mRNA messenger RNA
• cDNA complementary DNA
• pDNA plasmid DNA
• 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)).
• PNA peptide nucleic acids
• nucleic acid refers to any one or more nucleic acid segments, e.g.,
• 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.
• a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.
• the term "host cell” refers to a cell or a population of cells
• Host cells can be 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 NSO murine myeloma cell, a PER.C6® human cell, a Chinese hamster ovary (CHO) cell or a hybridoma).
• fungal cells ⁇ e.g., yeast cells such as Saccharomyces cerivisiae, Pichia pastoris, or Schizosaccharomyces pombe
• various animal cells such as insect cells ⁇ e.g., Sf-9) or mamm
• 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.
• references to a "substitution at position X" refer to the substitution of an amino acid present at position X with an alternative amino acid residue.
• substitution patterns can be 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.
• 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,
• 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,
• phenylalanine, methionine, tryptophan phenylalanine, methionine, tryptophan
• beta-branched side chains e.g., threonine, valine, isoleucine
• aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
• 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
• an electronegative residue e.g., Glu or Asp
• a hydrophilic residue e.g., Ser or Thr
• a hydrophobic residue e.g., Ala, Leu, He, Phe or Val
• a cysteine or proline is substituted for, or by, any other residue
• a residue having a bulky hydrophobic or aromatic side chain e.g., Val, His, He or Tip
• one having a smaller side chain e.g., Ala, Ser
• no side chain e.g., Gly
• substitutions can be readily identified by workers of ordinary skill.
• a substitution can be taken from any one of D-alanine, glycine, beta-alanine, L-cysteine and D-cysteine.
• a replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D- ornithine.
• 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.
• a polar residue e.g
• 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.
• insertion between 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.
• 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).
• B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
• BLASTN is used to compare nucleic acid sequences
• 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
• the members of the pair have the property of binding specifically to each other.
• 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.
• IgG 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 IgGl, IgG2, IgG3, and IgG4. In mice this class comprises IgGl, IgG2a, IgG2b, and IgG3.
• antigen binding domain describes the part of an antibody molecule which 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).
• 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.
• “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.
• 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.
• 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.
• chimeric antibody refers to antibodies wherein the amino acid
• sequence of the immunoglobulin molecule is derived from two or more species.
• the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of a mammal (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., MrkA) comprising a continuous or discontinuous site (i.e., an epitope) to which a complementary antibody specifically binds.
• 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
• the Kabat 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., Kabat et al, Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1 91)).
• amino acid position numbering as in Kabat
• Kabat position refers 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.
• 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.
• 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.
• EU index as in Kabat refers to the numbering system of the human
• Fc domain refers 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.
• an Fc domain contains the entire second constant domain CH2 (residues at EU positions 231-340 of human IgGl) and the third constant domain CH3 (residues at EU positions 341-447 of human IgGl).
• 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. Patent Nos. 5,624,821; 5,885,573; 5,677,425;
• sequences of the heavy chains of human IgGl, IgG2, IgG3 and IgG4 can be found in a number of sequence databases, for example, at the Uniprot database
• YTE refers to a set of mutations in an IgGl 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. Patent No.
• 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.
• 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.
• MrkA protein or fragments thereof may occur.
• Naturally occurring MrkA means MrkA protein which is naturally produced by a cell, without prior introduction of encoding nucleic acid using recombinant technology.
• naturally occurring MrkA may be as produced naturally by for example K. pneumoniae and/or as isolated from different members of the Klebsiella genus.
• Recombinant MrkA refers to a state in which the MrkA protein or fragments thereof may occur.
• Recombinant MrkA means MrkA protein or fragments thereof produced by recombinant DNA, e.g., in a heterologous host.
• Recombinant MrkA may differ from naturally occurring MrkA by glycosylation.
• 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.
• 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.
• subject refers to any animal (e.g., a mammal),
• subject and patient refer to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy of a condition associated with a Klebsiella infection.
• 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 Enterob acted aceae family. Klebsiella include, for example, K.
• an O antigen is a lipopolysaccharide
• the K antigen is a capsular polysaccharide.
• the structural variability of these antigens forms the basis for their classification into Klebsiella "serotypes.”
• the ability of a MrkA 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 O and/or K antigens.
• composition 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 a polypeptide e.g., an antigen binding protein
• an amount sufficient to carry out a specifically stated purpose 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.
• 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.
• the term "therapeutically effective” refers to an amount of a therapeutic agent that is capable of reducing MrkA 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.
• 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).
• a therapeutic agent e.g., an antigen binding protein including an antibody, as disclosed herein
• a therapeutic effect i.e., by administration of a therapeutically effective amount.
• such particular result is a reduction in MrkA activity in a patient in need thereof.
• label when used herein refers to a detectable compound
• 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.
• 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.
• 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.
• 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 t e Klebsiella -mediated disease or condition.
• An "immunologically effective amount" of a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or an immunogenic fragment thereof is an amount sufficient to enhance a subject's own immune response against Klebsiella.
• Levels of induced immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and/or serum antibodies, e.g., by complement fixation, enzyme-linked immunosorbent, serum bactericidal assay, opsonophagocytic killing assay, or biofilm formation inhibition assay.
• immunogenic fragment means a fragment that generates an immune response (i.e., has immunogenic activity) when administered, alone or optionally with a suitable adjuvant, to a subject.
• a "vaccine" composition according to the present invention is one comprising an immunogenically effective amount of MrkA, including immunogenically active truncates, portions, fragments and segments thereof, or a polynucleotide encoding MrkA, including immunogenically active truncates, portions, fragments and segments thereof and in any and all active combinations thereof, wherein said polypeptide, or active fragment, or fragments, or polynucleotides is/are suspended in a pharmacologically acceptable carrier, which includes all suitable diluents or excipients.
• an "immune response” refers to a response in the subject to the introduction of the MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding MrkA polypeptide or an immunogenic fragment thereof, generally characterized by, but not limited to, production of antibodies and/or T cells.
• an immune response may be a cellular response such as induction or activation of CD4+ T cells or CD8+ T cells or both, specific for Klebsiella, a humoral response of increased production of anti- Klebsiella antibodies, or both cellular and humoral responses.
• Immune responses can also include a mucosal response, e.g., a mucosal antibody response, e.g., S-IgA production or a mucosal cell-mediated response, e.g., T-cell response.
• a mucosal response e.g., a mucosal antibody response, e.g., S-IgA production or a mucosal cell-mediated response, e.g., T-cell response.
• a "protective immune response” refers to an immune response exhibited by a subject that is protective when the subject is exposed to Klebsiella. In some instances, the Klebsiella can still cause infection, but it cannot cause a serious infection. Typically, the protective immune response results in detectable levels of host engendered serum and antibodies that are capable of neutralizing Klebsiella in vitro and in vivo.
• adjuvant refers to any material having the ability to (1) alter or
• any compound which may increase the expression, antigenicity or immunogenicity of MrkA polypeptide or immunogenic fragment thereof provided herein is a potential adjuvant.
• 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 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, 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.
• the Klebsiella infection is a nosocomial infection.
• the Klebsiella infection is an opportunistic infection.
• the Klebsiella infection follows an organ transplant.
• 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
• 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
• HCDR3 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
• CDR3 can be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology.
• 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 110 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.
• 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.
• 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).
• MrkA binding molecules e.g., antibodies, antigen binding proteins, and antigen binding fragments thereof, that specifically bind MrkA, for example, Klebsiella MrkA.
• MrkA binding molecules e.g., antibodies, antigen binding proteins, and antigen binding fragments thereof specifically bind to K. pneumoniae MrkA.
• MrkA binding molecules are referred to herein interchangeably as "MrkA binding molecules", “MrkA binding proteins” or "MrkA binding agents”.
• K. pneumoniae MrkA refers to the amino acid sequence shown in Figure 2D (SEQ ID NO: 17).
• K. pneumoniae isolates commonly express two fimbrial adhesins, type 1 and type 3 fimbriae. The type 1 fimbriae are implicated in promoting K.
• Type 3 fimbriae mediate biofilm formation on biotic and abiotic surfaces and are required for mature biofilm development.
• the various components of type 3 fimbriae are encoded by the mrkABCDF operon, which produce the major pilin subunit MrkA, chaperone MrkB, outer membrane usher MrkC, adhesin MrkD and MrkF. See Yang et al. PLoS One. 2013 Nov 14;8(1 l):e79038.
• Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament.
• the type 3 fimbriae mediate binding to target tissue using the MrkD adhesin that is associated with the fimbrial shaft comprised of the MrkA protein. See Langstraat et al., Infect Immun. 2001 Sep; 69(9): 5805-5812. Host cell adherence and biofilm formation of Klebsiella are mediated by such MrkA pilins. See Chan et al., Langmuir 28: 7428-7435 (2012), which is herein incorporated by reference in its entirety.
• the disclosure provides an isolated antigen binding protein that is an antibody or polypeptide that specifically binds to MrkA.
• the antigen binding protein is an antigen binding fragment of an antibody that specifically binds to MrkA.
• the MrkA binding molecules are antibodies or
• the disclosure provides an isolated antigen binding protein thereof that is a murine, non-human, humanized, chimeric, resurfaced, or human antigen binding protein that specifically binds to MrkA.
• the MrkA binding molecules are humanized antibodies or antigen binding fragment thereof.
• the MrkA binding molecule is a human antibody or antigen binding fragment thereof.
• the disclosure provides an isolated antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) that specifically binds to MrkA, wherein said antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof): a) binds to at least two Klebsiella pneumoniae (K. pneumoniae) serotypes; b) induces opsonophagocytic killing (OPK) of K. pneumoniae or c) binds to at least two K. pneumoniae serotypes and induces OPK of K. pneumoniae.
• said antigen binding protein including, e.g., an anti-MrkA antibody or antigen binding fragment thereof
• the disclosure provides an isolated antigen binding protein that binds to at least two K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that binds to at least three K.
• the disclosure provides an isolated antigen binding protein that binds to at least four K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that binds to at least five K.
• the disclosure provides an isolated antigen binding protein that binds to at least six K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that binds to at least seven K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that binds to at least eight K.
• the disclosure provides an isolated antigen binding protein that binds to at least nine K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that binds to at least ten K.
• the disclosure provides an isolated antigen binding protein (e.g., an anti- MrkA antibody or antigen binding fragment thereof) that binds to at least one, two, three, four, five, six, seven, eight, nine, or ten of the serotypes of the K. pneumoniae listed in Table 5.
• an isolated antigen binding protein e.g., an anti- MrkA antibody or antigen binding fragment thereof
• the disclosure provides an isolated antigen binding protein that binds to the K. pneumoniae serotypes 01 :K2, 01 :K79, 02:K28, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, andO12:K80.
• the disclosure provides an isolated antigen binding protein (including, e.g., an anti-MrkA antibody or antigen binding fragment thereof) that induces OPK of Klebsiella, including e.g., K. pneumoniae.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least one K. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11,
• the disclosure provides an isolated antigen binding protein that induces OPK in at least two K. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, andO12:K80. In some embodiments, the disclosure provides an isolated antigen binding protein that induces OPK in at least three K.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least four T. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, andO12:K80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least four T. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least five K. pneumoniae serotypes selected from the group consisting of: 01 :K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, and O12:K80. In some embodiments, the disclosure provides an isolated antigen binding protein that induces OPK in at least six K.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least seven K. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least seven K. pneumoniae serotypes selected from the group consisting of: 01:K2, 01:K79, 02a:K28, 05:K57, 03:K58, 03:K11, 03:K25, 04:K15, 05:K61, 07: ⁇ 67, and ⁇ 12: ⁇ 80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least eight h, pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least nine K pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein that induces OPK in at least ten K pneumoniae serotypes selected from the group consisting of: 01 :K2, 01 :K79, 02a:K28, 05:K57, 03 :K58, 03 :K11, 03 :K25, 04:K15, 05:K61, 07:K67, and O12:K80.
• the disclosure provides an isolated antigen binding protein
• the disclosure provides an isolated antigen binding protein
• an anti-MrkA antibody or antigen binding fragment thereof that specifically binds to MrkA, wherein said antigen binding protein has at least one characteristic selected from the group consisting of: a) binds to at least two K pneumoniae serotypes; b) induces OPK of at least one or two K pneumoniae serotypes in vitro; c) reduces bacterial burden in a mouse Klebsiella infection model; and d) confers survival benefit in a mouse Klebsiella infection model.
• the disclosure provides an isolated antigen binding protein
• an anti-MrkA antibody or antigen binding fragment thereof that specifically binds to MrkA, wherein said antigen binding protein has at least two characteristics selected from the group consisting of: a) binds to at least two K pneumoniae serotypes; b) induces OPK of at least one or two K pneumoniae serotypes in vitro; c) reduces bacterial burden in a mouse Klebsiella infection model; and d) confers survival benefit in a mouse Klebsiella infection model.
• the disclosure provides an isolated antigen binding protein
• an anti-MrkA antibody or antigen binding fragment thereof that specifically binds to MrkA, wherein said antigen binding protein has at least three characteristic selected from the group consisting of: a) binds to at least two K pneumoniae serotypes; b) induces OPK of at least one or two K pneumoniae serotypes in vitro; c) reduces bacterial burden in a mouse Klebsiella infection model; and d) confers survival benefit in a mouse Klebsiella infection model.
• the disclosure provides an isolated antigen binding protein
• an anti-MrkA antibody or antigen binding fragment thereof that specifically binds to MrkA, wherein said antigen binding protein: a) binds to at least two K pneumoniae serotypes; b) induces OPK of at least one or two K pneumoniae serotypes in vitro; c) reduces bacterial burden in a mouse Klebsiella infection model; and d) confers survival benefit in a mouse Klebsiella infection model.
• MrkA-binding proteins disclosed herein include MrkA antibodies Kp3 and
• MrkA-binding proteins disclosed herein also include MrkA antibodies clone 1, clone 4, clone 5, and clone 6 and antigen-binding fragments thereof.
• the MrkA-binding proteins of the disclosure also include MrkA-binding proteins (e.g., anti-MrkA antibodies or antigen-binding fragments thereof) that specifically bind to the same MrkA epitope as Kp3 or Kpl6.
• the MrkA-binding proteins of the disclosure also include MrkA-binding proteins (e.g., anti-MrkA antibodies or antigen-binding fragments thereof) that specifically bind to the same MrkA epitope as clone 1, clone 4, clone 5, or clone 6.
• the disclosure provides an isolated antigen binding protein (e.g., anti-MrkA antibody or antigen-binding fragment thereof) that binds oligomeric MrkA.
• the antigen binding protein e.g., anti-MrkA antibody or antigen-binding fragment thereof
• the antigen binding protein binds to monomeric MrkA (e.g., clone 1, an antibody or antigen-binding fragment thereof that contains the six CDRs or the VH and VL of clone 1, or an antibody or antigen-binding fragment thereof that binds the same epitope as or competitively inhibits binding of clone 1 to MrkA).
• MrkA monomeric MrkA
• the antigen binding protein (including e.g., an anti-MrkA antibody or antigen-binding fragment thereof) binds to an epitope within amino acids 1-40 and 171-202 of SEQ ID NO: 17.
• the antigen binding protein (including e.g., an anti-MrkA antibody or antigen-binding fragment thereof) binds to the MrkA sequence set forth in SEQ ID NO: 17, but does not bind to MrkA lacking amino acids 1- 40 of SEQ ID NO: 17 (i.e., SEQ ID NO:26). In some embodiments, the antigen binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof) binds to the MrkA sequence set forth in SEQ ID NO: 17, but does not bind to MrkA lacking amino acids 171-202 of SEQ ID NO: 17 (i.e., SEQ ID NO:27).
• the antigen binding protein binds to the MrkA sequence set forth in SEQ ID NO: 17 but does not bind to MrkA lacking amino acids 1- 40 and 171-202 of SEQ ID NO: 17 (i.e., SEQ ID NO:28).
• the antigen binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof) specifically binds to MrkA (SEQ ID NO: 17), but does not bind to either SEQ ID NO:26 or SEQ ID NO:27.
• the antigen binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof) specifically binds to MrkA (SEQ ID NO: 17), but does not bind to any of SEQ ID NOs:26-28.
• MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding
• MrkA-binding protiens that competitively inhibit binding of Kp3 or Kpl6 to MrkA.
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• MrkA-binding protiens that competitively inhibit binding of clone 1, clone 4, clone 5, or clone 6 to MrkA.
• an anti- MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of Kp3 or Kpl6 to MrkA in a competition ELISA assay.
• an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of clone 1, clone 4, clone 5, or clone 6 to MrkA in a competition ELISA assay. In some embodiments, an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of Kp3 or Kpl6 to K. pneumoniae in a competition ELISA assay. In some embodiments, an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of clone 1, clone 4, clone 5, or clone 6 to K. pneumoniae in a competition ELISA assay. In some embodiments,
• an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of Kp3 or Kpl6 to K. pneumoniae strain 29011 in a competition ELISA assay. In some embodiments, an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of clone 1, clone 4, clone 5, or clone 6 to K. pneumoniae strain 29011 in a competition ELISA assay. In some embodiments, an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of Kp3, Kpl6, clone 1, clone 4, clone 5, or clone 6 to K.
• an anti-MrkA antibody or antigen-binding fragment thereof competitively inhibits binding of Kp3, Kpl6, clone 1, clone 4, clone 5, or clone 6 to K pneumoniae strain 985048 in a competition ELISA assay.
• 10 2 fold excess of the anti-MrkA antibody or antigen- binding fragment thereof decreases binding of 1 ⁇ g Kp3 to MrkA by at least 20% in a competitive ELISA assay. In some embodiments, 10 2 fold excess of the anti-MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to MrkA by at least 25% in a competitive ELISA assay. In some embodiments, 10 2 fold excess of the anti-MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to MrkA by at least 30%) in a competitive ELISA assay.
• 10 2 fold excess of the anti-MrkA antibody or antigen- binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K pneumoniae by at least 20%> in a competitive ELISA assay. In some embodiments, 10 2 fold excess of the anti-MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K
• 10 2 fold excess of the anti-MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K pneumoniae by at least 30%> in a competitive ELISA assay.
• 10 2 fold excess of the anti-MrkA antibody or antigen- binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K. pneumoniae strain 29011 by at least 20%) in a competitive ELISA assay. In some embodiments, 10 2 fold excess of the anti- MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K pneumoniae strain 29011 by at least 25% in a competitive ELISA assay. In some
• 10 2 fold excess of the anti-MrkA antibody or antigen-binding fragment thereof decreases binding of 1 ⁇ g Kp3 to K pneumoniae strain 29011 by at least 30%> in a competitive ELISA assay.
• the MrkA-binding proteins (including, e.g., anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation.
• the MrkA-binding proteins (including, e.g., anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 25%>. In some embodiments, the MrkA-binding proteins (e.g. anti- MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 30%. In some embodiments, the MrkA-binding proteins (e.g. anti- MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 40%. In some embodiments, the MrkA-binding proteins (e.g.
• the MrkA-binding proteins inhibit or reduce Klebsiella biofilm formation by at least 50%. In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 55%. In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 60%. In some embodiments, the MrkA-binding proteins (e.g. anti- MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by about 25% to about 65%. In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by about 50% to about 60%.
• the MrkA-binding proteins inhibit or reduce Klebsiella biofilm formation by at least 25%) at a concentration of about 3 ⁇ g/ml. In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 25% at a concentration of about 4 ⁇ g/ml. In some
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• the MrkA-binding proteins inhibit or reduce Klebsiella biofilm formation by at least 25% at a concentration of about 5 ⁇ g/ml.
• the MrkA-binding proteins inhibit or reduce Klebsiella biofilm formation by at least 50%) at a concentration of about 10 ⁇ g/ml. In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by at least 60% at a concentration of about 10 ⁇ g/ml.
• the MrkA-binding proteins inhibit or reduce Klebsiella biofilm formation by about 25%) to about 65% at a concentration of about 10 ⁇ g/ml. In some embodiments, the MrkA- binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella biofilm formation by about 50% to about 60% at a concentration of about 10 ⁇ igl ⁇ . [0190] In some embodiments, the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) inhibit or reduce Klebsiella cell adherence (e.g.,
• Klebsiella epithelial cell adherence The Klebsiella epithelial cell adherence.
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• Klebsiella cell adherence e.g.,
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• the MrkA-binding proteins inhibit or reduce Klebsiella cell adherence (e.g., Klebsiella epithelial cell adherence) by at least 30%.
• the MrkA-binding proteins inhibit or reduce Klebsiella cell adherence (e.g., Klebsiella epithelial cell adherence) by at least 40%.
• the MrkA-binding proteins e.g.
• anti-MrkA antibodies or antigen binding fragments thereof inhibit or reduce Klebsiella cell adherence (e.g., Klebsiella epithelial cell adherence) by about 20% to about 50%.
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• Klebsiella cell adherence e.g., Klebsiella epithelial cell adherence
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• Klebsiella cell adherence e.g.,
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• the MrkA-binding proteins inhibit or reduce Klebsiella cell adherence (e.g., Klebsiella epithelial cell adherence) by at least 30% at a concentration of about 10 ⁇ g/ml.
• the MrkA-binding proteins e.g.
• the MrkA-binding proteins inhibit or reduce Klebsiella cell adherence (e.g., Klebsiella epithelial cell adherence) by at least 40%) at a concentration of about 10 ⁇ g/ml.
• the MrkA-binding proteins e.g. anti-MrkA antibodies or antigen binding fragments thereof
• inhibit or reduce Klebsiella cell adherence e.g., Klebsiella epithelial cell adherence
• the MrkA-binding proteins e.g.
• the MrkA-binding proteins (e.g. anti-MrkA antibodies or antigen binding fragments thereof) also include MrkA-binding proteins that comprise the heavy and light chain complementarity determining region (CDR) sequences of Kp3, Kpl6, clone 1, clone 4, clone 5, or clone 6.
• CDR complementarity determining region
• Antigen binding proteins (including anti-MrkA 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-MrkA 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-MrkA Kp3, Kpl6, clone 1, clone 4, clone 5, and clone 6 antibodies are provided in Tables 3 and 4 below.
• HPWFDPWGQGTLVTVSS (SEQIDNO:56)
• the disclosure provides an isolated antigen binding protein
• the antigen binding protein comprises a heavy chain variable region (VH) at least 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 13-14 or 53-56 and a light chain variable region (VL) at least 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 15-16 or 57-60.
• the isolated antigen binding protein that specifically binds to MrkA comprises a heavy chain variable region comprising the sequences of SEQ ID NOs: 13-14 or 53-56 and a light chain variable region comprising the sequences of SEQ ID NOs: 15-16 or 57-60.
• the polypeptide having a certain percentage of sequence identity to SEQ ID NOs: 13-16 or 53-60 differs from SEQ ID NOs: 13-16 or 53-60 by conservative amino acid substitutions only.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 95% identical to SEQ ID NO: 13 and a VL at least 95% identical to SEQ ID NO: 15, a VH at least 95% identical to SEQ ID NO: 14 and a VL at least 95% identical to SEQ ID NO: 16, a VH at least 95% identical to SEQ ID NO:53 and a VL at least 95% identical to SEQ ID NO:57, a VH at least 95% identical to SEQ ID NO: 54 and a VL at least 95% identical to SEQ ID NO: 58, a VH at least 95% identical to SEQ ID NO:55 and a VL at least 95% identical to SEQ ID NO:59, or a VH at least 95% identical to SEQ ID NO:56 and a VL at least 95% identical to SEQ ID NO: 60, wherein the antigen binding protein binds to at least two K.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 95% identical to SEQ ID NO: 13 and a VL at least 95% identical to SEQ ID NO: 15, a VH at least 95% identical to SEQ ID NO: 14 and a VL at least 95% identical to SEQ ID NO: 16, a VH at least 95% identical to SEQ ID NO:53 and a VL at least 95% identical to SEQ ID NO:57, a VH at least 95% identical to SEQ ID NO: 54 and a VL at least 95% identical to SEQ ID NO: 58, a VH at least 95% identical to SEQ ID NO:55 and a VL at least 95% identical to SEQ ID NO:59, or a VH at least 95% identical to SEQ ID NO:56 and a VL at least 95% identical to SEQ ID NO: 60, wherein the antigen binding protein induces OPK of at least two K
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 95% identical to SEQ ID NO: 13 and a VL at least 95% identical to SEQ ID NO: 15, a VH at least 95% identical to SEQ ID NO: 14 and a VL at least 95% identical to SEQ ID NO: 16, a VH at least 95% identical to SEQ ID NO:53 and a VL at least 95% identical to SEQ ID NO:57, a VH at least 95% identical to SEQ ID NO: 54 and a VL at least 95% identical to SEQ ID NO: 58, a VH at least 95% identical to SEQ ID NO:55 and a VL at least 95% identical to SEQ ID NO:59, or a VH at least 95% identical to SEQ ID NO:56 and a VL at least 95% identical to SEQ ID NO: 60, wherein the antigen binding protein reduces bacterial burden in a subject
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 95% identical to SEQ ID NO: 13 and a VL at least 95% identical to SEQ ID NO: 15, a VH at least 95% identical to SEQ ID NO: 14 and a VL at least 95% identical to SEQ ID NO: 16, a VH at least 95% identical to SEQ ID NO:53 and a VL at least 95% identical to SEQ ID NO:57, a VH at least 95% identical to SEQ ID NO: 54 and a VL at least 95% identical to SEQ ID NO: 58, a VH at least 95% identical to SEQ ID NO:55 and a VL at least 95% identical to SEQ ID NO:59, or a VH at least 95% identical to SEQ ID NO:56 and a VL at least 95% identical to SEQ ID NO: 60, wherein the antigen binding protein confers survival benefit in a subject.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 96% identical to SEQ ID NO: 13 and a VL at least 96% identical to SEQ ID NO: 15, a VH at least 96% identical to SEQ ID NO: 14 and a VL at least 96% identical to SEQ ID NO: 16, a VH at least 96% identical to SEQ ID NO:53 and a VL at least 96% identical to SEQ ID NO:57, a VH at least 96% identical to SEQ ID NO: 54 and a VL at least 96% identical to SEQ ID NO: 58, a VH at least 96% identical to SEQ ID NO:55 and a VL at least 96% identical to SEQ ID NO:59, or a VH at least 96% identical to SEQ ID NO:56 and a VL at least 96% identical to SEQ ID NO: 60, wherein the antigen binding protein binds to at least two K.
• said antigen binding protein comprises a VH at least 96% identical to SEQ ID NO: 13 and a VL at least 96% identical to SEQ ID NO: 15, a VH at least 96% identical to SEQ ID NO: 14 and a VL at least 96% identical to SEQ ID NO: 16, a VH at least 96% identical to SEQ ID NO:53 and a VL at least 96% identical to SEQ ID NO:57, a VH at least 96% identical to SEQ ID NO: 54 and a VL at least 96% identical to SEQ ID NO: 58, a VH at least 96% identical to SEQ ID NO:55 and a VL at least 96% identical to SEQ ID NO:59, or a VH at least 96% identical to SEQ ID NO:56 and a VL at least 96% identical to SEQ ID NO: 60, wherein the antigen binding protein induces OPK of at least two K
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 96% identical to SEQ ID NO: 13 and a VL at least 96% identical to SEQ ID NO: 15, a VH at least 96% identical to SEQ ID NO: 14 and a VL at least 96% identical to SEQ ID NO: 16, a VH at least 96% identical to SEQ ID NO:53 and a VL at least 96% identical to SEQ ID NO:57, a VH at least 96% identical to SEQ ID NO: 54 and a VL at least 96% identical to SEQ ID NO: 58, a VH at least 96% identical to SEQ ID NO:55 and a VL at least 96% identical to SEQ ID NO:59, or a VH at least 96% identical to SEQ ID NO:56 and a VL at least 96% identical to SEQ ID NO: 60, wherein the antigen binding protein reduces bacterial burden in a subject
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 96% identical to SEQ ID NO: 13 and a VL at least 96% identical to SEQ ID NO: 15, a VH at least 96% identical to SEQ ID NO: 14 and a VL at least 96% identical to SEQ ID NO: 16, a VH at least 96% identical to SEQ ID NO:53 and a VL at least 96% identical to SEQ ID NO:57, a VH at least 96% identical to SEQ ID NO: 54 and a VL at least 96% identical to SEQ ID NO: 58, a VH at least 96% identical to SEQ ID NO:55 and a VL at least 96% identical to SEQ ID NO:59, or a VH at least 96% identical to SEQ ID NO:56 and a VL at least 96% identical to SEQ ID NO: 60, wherein the antigen binding protein confers survival benefit in a subject.
• said antigen binding protein comprises a VH at least 97% identical to SEQ ID NO: 13 and a VL at least 97% identical to SEQ ID NO: 15, a VH at least 97% identical to SEQ ID NO: 14 and a VL at least 97% identical to SEQ ID NO: 16, a VH at least 97% identical to SEQ ID NO:53 and a VL at least 97% identical to SEQ ID NO:57, a VH at least 97% identical to SEQ ID NO: 54 and a VL at least 97% identical to SEQ ID NO: 58, a VH at least 97% identical to SEQ ID NO:55 and a VL at least 97% identical to SEQ ID NO:59, or a VH at least 97% identical to SEQ ID NO:56 and a VL at least 97% identical to SEQ ID NO: 60, wherein the antigen binding protein binds to at least two K.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 97% identical to SEQ ID NO: 13 and a VL at least 97% identical to SEQ ID NO: 15, a VH at least 97% identical to SEQ ID NO: 14 and a VL at least 97% identical to SEQ ID NO: 16, a VH at least 97% identical to SEQ ID NO:53 and a VL at least 97% identical to SEQ ID NO:57, a VH at least 97% identical to SEQ ID NO: 54 and a VL at least 97% identical to SEQ ID NO: 58, a VH at least 97% identical to SEQ ID NO:55 and a VL at least 97% identical to SEQ ID NO:59, or a VH at least 97% identical to SEQ ID NO:56 and a VL at least 97% identical to SEQ ID NO: 60, wherein the antigen binding protein induces OPK of at least two K
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 97% identical to SEQ ID NO: 13 and a VL at least 97% identical to SEQ ID NO: 15, a VH at least 97% identical to SEQ ID NO: 14 and a VL at least 97% identical to SEQ ID NO: 16, a VH at least 97% identical to SEQ ID NO:53 and a VL at least 97% identical to SEQ ID NO:57, a VH at least 97% identical to SEQ ID NO: 54 and a VL at least 97% identical to SEQ ID NO: 58, a VH at least 97% identical to SEQ ID NO:55 and a VL at least 97% identical to SEQ ID NO:59, or a VH at least 97% identical to SEQ ID NO:56 and a VL at least 97% identical to SEQ ID NO: 60, wherein the antigen binding protein reduces bacterial burden in a subject
• said antigen binding protein comprises a VH at least 97% identical to SEQ ID NO: 13 and a VL at least 97% identical to SEQ ID NO: 15, a VH at least 97% identical to SEQ ID NO: 14 and a VL at least 97% identical to SEQ ID NO: 16, a VH at least 97% identical to SEQ ID NO:53 and a VL at least 97% identical to SEQ ID NO:57, a VH at least 97% identical to SEQ ID NO: 54 and a VL at least 97% identical to SEQ ID NO: 58, a VH at least 97% identical to SEQ ID NO:55 and a VL at least 97% identical to SEQ ID NO:59, or a VH at least 97% identical to SEQ ID NO:56 and a VL at least 97% identical to SEQ ID NO: 60, wherein the antigen binding protein confers survival benefit in a subject.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 98% identical to SEQ ID NO: 13 and a VL at least 98% identical to SEQ ID NO: 15, a VH at least 98% identical to SEQ ID NO: 14 and a VL at least 98% identical to SEQ ID NO: 16, a VH at least 98% identical to SEQ ID NO:53 and a VL at least 98% identical to SEQ ID NO:57, a VH at least 98% identical to SEQ ID NO: 54 and a VL at least 98% identical to SEQ ID NO: 58, a VH at least 98% identical to SEQ ID NO:55 and a VL at least 98% identical to SEQ ID NO:59, or a VH at least 98% identical to SEQ ID NO:56 and a VL at least 98% identical to SEQ ID NO: 60, wherein the antigen binding protein binds to at least two K.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 98% identical to SEQ ID NO: 13 and a VL at least 98% identical to SEQ ID NO: 15, a VH at least 98% identical to SEQ ID NO: 14 and a VL at least 98% identical to SEQ ID NO: 16, a VH at least 98% identical to SEQ ID NO:53 and a VL at least 98% identical to SEQ ID NO:57, a VH at least 98% identical to SEQ ID NO: 54 and a VL at least 98% identical to SEQ ID NO: 58, a VH at least 98% identical to SEQ ID NO:55 and a VL at least 98% identical to SEQ ID NO:59, or a VH at least 98% identical to SEQ ID NO:56 and a VL at least 98% identical to SEQ ID NO: 60, wherein the antigen binding protein induces OPK of at least two K
• said antigen binding protein comprises a VH at least 98% identical to SEQ ID NO: 13 and a VL at least 98% identical to SEQ ID NO: 15, a VH at least 98% identical to SEQ ID NO: 14 and a VL at least 98% identical to SEQ ID NO: 16, a VH at least 98% identical to SEQ ID NO:53 and a VL at least 98% identical to SEQ ID NO:57, a VH at least 98% identical to SEQ ID NO: 54 and a VL at least 98% identical to SEQ ID NO: 58, a VH at least 98% identical to SEQ ID NO:55 and a VL at least 98% identical to SEQ ID NO:59, or a VH at least 98% identical to SEQ ID NO:56 and a VL at least 98% identical to SEQ ID NO: 60, wherein the antigen binding protein reduces bacterial burden in a subject
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 98% identical to SEQ ID NO: 13 and a VL at least 98% identical to SEQ ID NO: 15, a VH at least 98% identical to SEQ ID NO: 14 and a VL at least 98% identical to SEQ ID NO: 16, a VH at least 98% identical to SEQ ID NO:53 and a VL at least 98% identical to SEQ ID NO:57, a VH at least 98% identical to SEQ ID NO: 54 and a VL at least 98% identical to SEQ ID NO: 58, a VH at least 98% identical to SEQ ID NO:55 and a VL at least 98% identical to SEQ ID NO:59, or a VH at least 98% identical to SEQ ID NO:56 and a VL at least 98% identical to SEQ ID NO: 60, wherein the antigen binding protein confers survival benefit in a subject.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 99% identical to SEQ ID NO: 13 and a VL at least 99% identical to SEQ ID NO: 15, a VH at least 99% identical to SEQ ID NO: 14 and a VL at least 99% identical to SEQ ID NO: 16, a VH at least 99% identical to SEQ ID NO:53 and a VL at least 99% identical to SEQ ID NO:57, a VH at least 99% identical to SEQ ID NO: 54 and a VL at least 99% identical to SEQ ID NO: 58, a VH at least 99% identical to SEQ ID NO:55 and a VL at least 99% identical to SEQ ID NO:59, or a VH at least 99% identical to SEQ ID NO:56 and a VL at least 99% identical to SEQ ID NO: 60, wherein the antigen binding protein binds to at least two K.
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 99% identical to SEQ ID NO: 13 and a VL at least 99% identical to SEQ ID NO: 15, a VH at least 99% identical to SEQ ID NO: 14 and a VL at least 99% identical to SEQ ID NO: 16, a VH at least 99% identical to SEQ ID NO:53 and a VL at least 99% identical to SEQ ID NO:57, a VH at least 99% identical to SEQ ID NO: 54 and a VL at least 99% identical to SEQ ID NO: 58, a VH at least 99% identical to SEQ ID NO:55 and a VL at least 99% identical to SEQ ID NO:59, or a VH at least 99% identical to SEQ ID NO:56 and a VL at least 99% identical to SEQ ID NO: 60, wherein the antigen binding protein induces OPK of at least two K
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 99% identical to SEQ ID NO: 13 and a VL at least 99% identical to SEQ ID NO: 15, a VH at least 99% identical to SEQ ID NO: 14 and a VL at least 99% identical to SEQ ID NO: 16, a VH at least 99% identical to SEQ ID NO:53 and a VL at least 99% identical to SEQ ID NO:57, a VH at least 99% identical to SEQ ID NO: 54 and a VL at least 99% identical to SEQ ID NO: 58, a VH at least 99% identical to SEQ ID NO:55 and a VL at least 99% identical to SEQ ID NO:59, or a VH at least 99% identical to SEQ ID NO:56 and a VL at least 99% identical to SEQ ID NO: 60, wherein the antigen binding protein reduces bacterial burden in a subject
• the disclosure provides an isolated antigen binding protein
• said antigen binding protein comprises a VH at least 99% identical to SEQ ID NO: 13 and a VL at least 99% identical to SEQ ID NO: 15, a VH at least 99% identical to SEQ ID NO: 14 and a VL at least 99% identical to SEQ ID NO: 16, a VH at least 99% identical to SEQ ID NO:53 and a VL at least 99% identical to SEQ ID NO:57, a VH at least 99% identical to SEQ ID NO: 54 and a VL at least 99% identical to SEQ ID NO: 58, a VH at least 99% identical to SEQ ID NO:55 and a VL at least 99% identical to SEQ ID NO:59, or a VH at least 99% identical to SEQ ID NO:56 and a VL at least 99% identical to SEQ ID NO: 60, wherein the antigen binding protein confers survival benefit in a subject.
• 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
• ELISA immunosorbent assay
• monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Patent 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.
• monoclonal antibodies are generated by the host cells.
• 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.
• 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.
• 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.
• the monoclonal antibody against the MrkA is a humanized antibody.
• 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.
• the antibody to MrkA 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 (l):86-95; and U.S. Patent 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.
• 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. Patents 5,545,807; 5,545,806; 5,569,825; 5,625, 126; 5,633,425; and 5,661,016.
• 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 molecule; (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 molecule with papain and a reducing agent, and (d) Fv fragments.
• Antigen binding proteins of the present disclosure can further comprise antibody constant regions or parts thereof.
• a VL domain can be attached at its C-terminal end to antibody light chain constant domains including human CK or Cy chains.
• an antigen binding protein based on a VH domain can be attached at its C-terminal end to all or part (e.g. a CHI 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 IgGl and IgG4.
• the immunoglobulin heavy chain can be derived from the antibody isotype sub-class, IgGl .
• 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:
• 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).
• ADCC antigen-dependent cell-mediated cytotoxicity
• CDC complement dependent cytotoxicity
• 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
• pharmacokinetic properties e.g., half-life
• L234F/L235E/P331 S (“TM") causes a profound decrease in the binding activity of human IgGl molecules to human Clq, 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.
• the antigen-binding protein 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 IgGl constant domain; (e) an IgG2 constant domain; (f) an IgG3 constant domain; (g) an IgG4 constant domain; and (h) an IgM constant domain.
• the antigen-binging protein is an antibody or an antigen-binding fragment thereof that comprises an IgGl heavy chain immunoglobulin constant domain.
• the antigen-binding protein is an antibody or an antigen-binding fragment thereof that comprises an IgGl/IgG3 chimeric heavy chain immunoglobulin constant domain.
• the antigen-binding protein of the disclosure 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 can further comprise a human IgGl constant domain and a human lambda constant domain.
• the antigen-binding protein of the disclosure can comprise an IgG Fc domain containing a mutation at positions 252, 254 and 256, wherein the position numbering is according to the EU index as in Kabat.
• the IgGl 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 anti-MrkA antibodies or antigen binding
• 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.
• isotopic labels such as biotin signaling peptides, Green Fluorescent Proteins (GFPs), blue fluorescent proteins (BFPs), cyan fluorescent proteins (CFPs), and yellow fluorescent proteins (YFPs)
• the disclosure also provides a pharmaceutical composition
• a pharmaceutical composition comprising one or more of the MrkA-binding agents (including, e.g., anti-MrkA antibodies or antigen binding fragments) described herein, a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or an immunogenic fragment thereof.
• the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle or pharmaceutically acceptable excipient.
• these pharmaceutical compositions find use in treating, preventing or ameliorating a condition associated with a Klebsiella infection in human patients. In certain embodiments, these pharmaceutical compositions find use in inhibiting growth of Klebsiella.
• formulations are prepared for storage and use by
• compositions of the present disclosure can be administered in any number of ways for either local or systemic treatment.
• a pharmaceutical composition comprising one or more of the MrkA-binding agents (including, e.g., anti-MrkA antibodies or antigen binding fragments), MrkA polypeptides, immunogenic fragments thereof, or polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof described herein is used for treating pneumonia, urinary tract infection, septicemia, neonatal septicemia, 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.
• the MrkA-binding agents including, e.g., anti-MrkA antibodies or antigen binding fragments
• MrkA polypeptides including, e.g., anti-MrkA antibodies or antigen binding fragments
• MrkA polypeptides including,
• a pharmaceutical composition comprising one or more of the MrkA-binding agents (including, e.g., anti-MrkA antibodies or antigen binding fragments), MrkA polypeptides, immunogenic fragments thereof, or polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof 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, and/or K. granulomatis).
• MrkA-binding agents including, e.g., anti-MrkA antibodies or antigen binding fragments
• MrkA polypeptides including, e.g., anti-MrkA antibodies or antigen binding fragments
• polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof described herein is useful in nosocomial infections,
• a pharmaceutical composition comprising one or more of the MrkA-binding agents (including, e.g., anti-MrkA antibodies or antigen binding fragments), MrkA polypeptides, immunogenic fragments thereof, or polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof described herein is useful in subjects exposed to ⁇ Klebsiella contaminated device, including, e.g., a ventilator, a catheter, or an intravenous catheter.
• the MrkA-binding agents including, e.g., anti-MrkA antibodies or antigen binding fragments
• MrkA polypeptides including, e.g., anti-MrkA antibodies or antigen binding fragments
• polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof described herein is useful in subjects exposed to ⁇ Klebsiella contaminated device, including, e.g., a ventilator, a catheter, or an intravenous catheter.
• the pharmaceutical composition comprises an amount of a
• MrkA-binding agent e.g., an antibody or antigen-binding fragment thereof
• the Klebsiella is K. pneumoniae, K. oxytoca, K. planticola, and/or K. granulomatis.
• the Klebsiella is K. pneumoniae, K. oxytoca, and/or K. granulomatis.
• the Klebsiella is K. pneumoniae.
• the pharmaceutical composition comprises an amount of a
• the Klebsiella is K pneumoniae, K oxytoca, K planticola, 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.
• the methods of treating, preventing and/or ameliorating a condition associated with & Klebsiella infection comprises contacting a subject infected with a Klebsiella with a pharmaceutical composition comprising a MrkA-binding protien ⁇ e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof in vivo.
• a MrkA-binding protien e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• a MrkA polypeptide an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof in vivo.
• a pharmaceutical composition comprising a MrkA-binding protein, a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered at the same time or shortly after a subject has been exposed to bacteria to prevent infection.
• the pharmaceutical composition comprising a MrkA-binding protein is administered as a therapeutic after infection.
• Klebsiella infections comprises administering to a subject a pharmaceutical composition comprising a MrkA-binding agent ⁇ e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof.
• a pharmaceutical composition comprising a MrkA-binding agent ⁇ e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof.
• the subject is a human.
• the pharmaceutical composition comprising a MrkA-binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered before the subject is infected with Klebsiella.
• a MrkA-binding protein e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• a MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered before the subject is infected with Klebsiella.
• the pharmaceutical composition comprising a MrkA- binding protein (e.g., an-anti MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered after the subject is infected with a Klebsiella.
• a MrkA- binding protein e.g., an-anti MrkA antibody or antigen-binding fragment thereof
• a MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered after the subject is infected with a Klebsiella.
• the pharmaceutical composition comprising a MrkA- binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered to a subject on a ventilator.
• a catheter e.g., a urinary catheter or an intravenous catheter.
• the subject is receiving antibiotics.
• a pharmaceutical composition comprising a MrkA- binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), a MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a nosocomial Klebsiella infection.
• a MrkA- binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• a MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a nosocomial Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an opportunistic Klebsiella infection.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an opportunistic Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a Klebsiella infection following an organ transplant.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a Klebsiella infection following an organ transplant.
• a pharmaceutical composition comprising a MrkA- binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin resistant Klebsiella infection.
• a MrkA- binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen- binding fragment thereof) MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an aminoglycoside resistant Klebsiella infection.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen- binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an aminoglycoside resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti- MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a quinolone resistant Klebsiella infection.
• a MrkA-binding agent e.g., an anti- MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a quinolone resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA- binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a carbapenem resistant Klebsiella infection.
• a MrkA- binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a carbapenem resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen- binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin, aminoglycoside, quinolone, and carbapenem resistant Klebsiella infection.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen- binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin, aminoglycoside, quinolone, and carbapenem resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of infection with Klebsiella that produce extended spectrum beta-lactamase (ESBL).
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of infection with Klebsiella that produce extended spectrum beta-lactamase (ESBL).
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti- MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin, aminoglycoside, and quinolone resistant Klebsiella infection.
• a MrkA-binding agent e.g., an anti- MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of a cephalosporin, aminoglycoside, and quinolone resistant Klebsiella infection.
• a pharmaceutical composition comprising a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen- binding fragment thereof), MrkA polypeptide, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an infection with Klebsiella that produce carbapenemase.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen- binding fragment thereof
• MrkA polypeptide e.g., an immunogenic fragment thereof
• a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is for the treatment or prevention of an infection with Klebsiella that produce carbapenemase.
• Klebsiella infection the appropriate dosage of a pharmaceutical composition, antibody, anti- MrkA binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof 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, anti-MrkA binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof 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, anti-MrkA binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof 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.
• MrkA, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof can be administered to a subject to protect from infection with Klebsiella, e.g., by eliciting antibodies to a protective MrkA antigen.
• an immunogenic composition comprising MrkA, an immunogenic fragment thereof, or a polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof can be utilized to produce antibodies to diagnose Klebsiella infections, or to produce vaccines for prophylaxis and/or treatment of such Klebsiella infections as well as booster vaccines to maintain a high titer of antibodies against the immunogen(s) of the immunogenic composition.
• the MrkA or immunogenic fragment thereof is K.
• the MrkA or immunogenic fragment thereof is K. pneumoniae MrkA. In some embodiments, the MrkA or immunogenic fragment thereof comprises the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof is monomelic. In some embodiments, the MrkA or immunogenic fragment thereof is oligomeric.
• the MrkA or immunogenic fragment thereof comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 80%) identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 85%> identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 17.
• the MrkA or immunogenic fragment thereof comprises a sequence at least 96%) identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 97% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 98% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 99% identical to the sequence set forth in SEQ ID NO: 17.
• the MrkA or immunogenic fragment thereof comprises amino acids 1-40 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-50 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-100 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises amino acids 1- 150 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-175 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises amino acids 171-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 150-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some
• the MrkA or immunogenic fragment thereof comprises amino acids 100-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 50-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises amino acids 1-40 and 171-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 85%) identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 19.
• the MrkA or immunogenic fragment thereof comprises a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 96% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 97%) identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 98% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the MrkA or immunogenic fragment thereof comprises a sequence at least 99% identical to the sequence set forth in SEQ ID NO: 19.
• the MrkA or immunogenic fragment thereof comprises amino acids 1-42 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-50 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-100 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises amino acids 1- 150 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 1-175 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the MrkA or immunogenic fragment thereof comprises amino acids 173-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 150-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some
• the MrkA or immunogenic fragment thereof comprises amino acids 100-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the MrkA or immunogenic fragment thereof comprises amino acids 50-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• Vaccines can be prepared as injectables, either as liquid solutions or suspensions.
• Vaccines in an oil base are also well known such as for inhaling. Solid forms which are dissolved or suspended prior to use can also be formulated.
• Pharmaceutical carriers, diluents and excipients are generally added that are compatible with the active ingredients and acceptable for pharmaceutical use. Examples of such carriers include, but are not limited to, water, saline solutions, dextrose, or glycerol. Combinations of carriers may also be used.
• Vaccine compositions can comprise substances to stabilize pH, or to function as adjuvants, wetting agents, or emulsifying agents, which can serve to improve the effectiveness of the vaccine.
• a vaccine comprises one or more adjuvants.
• Vaccine administration is generally by conventional routes, for instance,
• intravenous, subcutaneous, intraperitoneal, or mucosal routes can be by parenteral injection, for example, a subcutaneous or intramuscular injection.
• the vaccine may be given in a single dose schedule, or optionally in a multiple dose schedule.
• the amount of vaccine sufficient to confer immunity to Klebsiella is determined by methods well known to those skilled in the art. This quantity will be determined based upon the characteristics of the vaccine recipient, including considerations of age, sex, and general physical condition, and the level of immunity required.
• MrkA-binding agents including, e.g., anti-MrkA antibodies and antigen- binding fragments thereof), MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof described herein are useful in a variety of applications including, but not limited to, pneumonia, urinary tract infection, septicemia, neonatal septicemia, 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.
• PHA pyogenic liver abscesses
• the MrkA-binding agents include antibodies and antigen-binding fragments thereof), MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic 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, and/or K. granulomatis).
• the MrkA-binding agents, MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding MrkA polypeptides or immunogenic fragments thereof are useful in subjects exposed to & Klebsiella contaminated device, including, e.g., a ventilator, a catheter, or an intravenous catheter.
• the disclosure provides methods of treating, preventing and/or ameliorating a condition associated with & Klebsiella infection comprising
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof to a subject.
• the amount is effective to inhibit growth of the Klebsiella in the subject.
• the Klebsiella is K. pneumoniae, K. oxytoca, K. planticola, and/or K. granulomatis.
• the Klebsiella is K. pneumoniae, K. oxytoca, and/or K. granulomatis.
• the Klebsiella is K. pneumoniae.
• the subject has been exposed to Klebsiella.
• Klebsiella has been detected in the subject.
• the subject is suspected of being infected with Klebsiella, e.g., based on symptoms.
• the disclosure provides methods of treating, preventing and/or ameliorating a condition associated with & Klebsiella infection comprising
• the Klebsiella is K.
• the Klebsiella is K. pneumoniae, K. oxytoca, and/or K. granulomatis. In some embodiments, the Klebsiella is K. pneumoniae. 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.
• the disclosure further provides methods of inhibiting growth of Klebsiella comprising administering a MrkA-binding agent (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof to a subject.
• a MrkA-binding agent e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• the Klebsiella is K pneumoniae, K oxytoca, K. planticola, and/or K granulomatis .
• the Klebsiella is K pneumoniae, K oxytoca, and/or K granulomatis.
• the Klebsiella is K pneumoniae.
• 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.
• the methods of treating, preventing and/or ameliorating a condition associated with & Klebsiella infection comprises contacting a subject infected with a Klebsiella with the MrkA-binding agent ⁇ e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof in vivo.
• contacting a cell with a MrkA-binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is undertaken in a subject.
• MrkA-binding agents for example, MrkA-binding agents, MrkA polypeptides, immunogenic fragments thereof, and polynucleotides encoding a MrkA polypeptides or immunogenic fragments thereof can be administered to a mouse Klebsiella infection model to reduce bacterial burden.
• the MrkA-binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered before introduction of bacteria to the subject to prevent infections.
• the MrkA-binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered at the same time or shortly after the subject has been exposed to bacteria to prevent infection. In some embodiments, the MrkA-binding agent, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered to the subject as a therapeutic after infection.
• Klebsiella infections comprises administering to a subject an effective amount of a MrkA- binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof.
• the subject is a human.
• the effective amount of a MrkA-binding protein e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered before the subject or patient is infected with Klebsiella.
• the effective amount of a MrkA-binding protein e.g., an anti-MrkA antibody or antigen- binding fragment thereof
• MrkA polypeptide, immunogenic fragment thereof or
• polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof is administered after the subject or patient is infected with a Klebsiella.
• the subject is on a ventilator.
• the subject has a catheter (e.g., a urinary catheter or an intravenous catheter).
• the subject is receiving antibiotics.
• 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.
• the Klebsiella is cephalosporin resistant. In certain embodiments, the Klebsiella is cephalosporin resistant. In certain
• 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 produce extended spectrum beta-lactamase (ESBL). In certain embodiments, the Klebsiella is cephalosporin, aminoglycoside, and quinolone resistant. In certain embodiments, the Klebsiella produce carbapenemase.
• ESBL beta-lactamase
• Klebsiella infections comprises administering to a subject an effective amount of a MrkA- binding protein (e.g., an anti-MrkA antibody or antigen-binding fragment thereof), MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA
• a MrkA- binding protein e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• immunogenic fragment thereof e.g., immunogenic fragment thereof, or polynucleotide encoding a MrkA
• the MrkA-binding protien e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof and the antibiotic can be administered simultaneously or sequentially.
• the MrkA-binding protien e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof and the antibiotic can be administered in the same pharmaceutical composition.
• the MrkA-binding protein e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• MrkA polypeptide e.g., an anti-MrkA antibody or antigen-binding fragment thereof
• the antibiotic can be administered in separate pharmaceutical compositions simultaneously or sequentially.
• the antibiotic can be, for example, a carbapanem or colistin.
• the present disclosure also provides methods of detecting MrkA, e.g., MrkA
• a method of detecting MrkA or a MrkA oligomer comprises contacting a sample with a MrkA antibody or antigen-binding fragment thereof provided herein and assaying for binding of the antibody or antigen-binding fragment thereof to the sample. Methods of assessing binding are well known in the art.
• kits comprising an isolated antigen-binding protein (e.g. an anti-MrkA antibody molecule or antigen-binding fragment thereof), MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof according to any aspect or embodiment of the present disclosure is also provided as an aspect of the present disclosure.
• the antigen-binding protein or anti-MrkA antibody, MrkA polypeptide, immunogenic fragment thereof, or polynucleotide encoding a MrkA polypeptide or immunogenic fragment thereof can be labeled to allow its reactivity in a sample to be determined, e.g. as described further below.
• Kits can be employed in diagnostic analysis or other methods for which antibody molecules 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.
• kits of the disclosure Ancillary materials to assist in or to enable performing such a method may be included within a kit of the disclosure.
• 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 MrkA in a sample by employing an antigen-binding protein provided by the present disclosure in a competition assay.
• the physical separation of bound from unbound antigen is not required.
• 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.
• 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 MrkA directly, by employing an antigen-binding protein according to the disclosure. In some embodiments, these methods utilize a biosensor system.
• the present disclosure provides an isolated nucleic acid
• the present disclosure provides methods of making or preparing an antigen-binding protein, a VH domain and/or a VL domain, MrkA polypeptide, or immunogenic fragment thereof described herein, comprising expressing said nucleic acid under conditions to bring about production of said antigen-binding protein, VH domain and/or VL domain, MrkA polypeptide, or immunogenic fragment thereof and, optionally, recovering the antigen-binding protein, VH domain and/or VL domain, MrkA polypeptide, or immunogenic fragment thereof.
• a nucleic acid provided by the present disclosure includes DNA and/or RNA.
• the nucleic acid is cDNA.
• 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 molecule, e.g., scFv or IgGl, as described above.
• 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.
• the VH CDR is selected from SEQ ID NOs: 1-6 or 29-40.
• the VL CDR is selected from SEQ ID NOs: 7-12 or 41-52.
• a nucleic acid encoding the Kp3, Kpl6, clone 1, clone 4, clone 5, or clone 6 set of CDRs, a nucleic acid encoding the Kp3, Kpl6, clone 1, clone 4, clone 5, or clone 6 set of HCDRs and a nucleic acid encoding the Kp3, KP16, clone 1, clone 4, clone 5, or clone 6 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.
• the nucleic acids of the present disclosure encode a VH and/or VL domain of Kp3, Kpl6, clone 1, clone 4, clone 5, or clo
• the polynucleotide encodes a sequence at least 75%
• polynucleotide encodes a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 96% identical to the sequence set forth in SEQ ID NO: 17.
• the polynucleotide encodes a sequence at least 97% identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 98%) identical to the sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide encodes a sequence at least 99% identical to the sequence set forth in SEQ ID NO: 17.
• the polynucleotide encodes amino acids 1-40 of SEQ ID NO:
• polynucleotide encodes amino acids 1-50 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polynucleotide encodes amino acids 1-100 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 1-150 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polynucleotide encodes amino acids 1-175 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 171-202 of SEQ
• the polynucleotide encodes amino acids 150-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polynucleotide encodes amino acids 100-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 50-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. [0279] In some embodiments, the polynucleotide encodes amino acids 1-40 and 171-202 of SEQ ID NO: 17 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes the sequence set forth in SEQ
• the polynucleotide encodes a sequence at least 75% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the
• polynucleotide encodes a sequence at least 80% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 85% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 90% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 96% identical to the sequence set forth in SEQ ID NO: 19.
• the polynucleotide encodes a sequence at least 97% identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 98%) identical to the sequence set forth in SEQ ID NO: 19. In some embodiments, the polynucleotide encodes a sequence at least 99% identical to the sequence set forth in SEQ ID NO: 19.
• the polynucleotide encodes amino acids 1-42 of SEQ ID NO:
• polynucleotide encodes amino acids 1-50 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polynucleotide encodes amino acids 1-100 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 1-150 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto. In some embodiments, the polynucleotide encodes amino acids 1-175 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 173-204 of SEQ
• the polynucleotide encodes amino acids 150-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 100-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• the polynucleotide encodes amino acids 50-204 of SEQ ID NO: 19 or a sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical thereto.
• 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.
• the present disclosure also provides a recombinant host cell which comprises one or more nucleic acids, plasmids, vectors or as described above.
• 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, MrkA polypeptide, or immunogenic fragment thereof can be isolated and/or purified using any suitable technique.
• the host cell is a mammalian host cell, such as a NS0 murine myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell.
• a mammalian host cell such as a NS0 murine myeloma cell, a PER.C6® human cell, or a Chinese hamster ovary (CHO) cell.
• Antigen-binding proteins VH and/or VL domains, MrkA polypeptides,
• nucleic acids according to the present disclosure may comprise DNA or RNA and can be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein
• 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, NSO 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.
• E. coli is well established in the art. For a review, see for example Pliickthun, A.
• 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.
• plasmids viral e.g. 'phage, or phagemid, as appropriate.
• 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.
• a further aspect of the present disclosure provides a host cell containing nucleic acid as disclosed herein.
• the disclosure provides a host cell transformed with nucleic acid comprising a nucleotide sequence encoding an antigen-binding protein of the present disclosure or antibody CDR, set of CDRs, VH and/or VL domain of an antigen- binding protein, MrkA polypeptide, or immunogenic fragment thereof of the present disclosure.
• the host cell comprises the expressed antigen-binding protein of the present disclosure or antibody CDR, set of CDRs, VH and/or VL domain of an antigen-binding protein, MrkA polypeptide, or immunogenic fragment thereof of the present disclosure.
• 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.
• 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 be 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.
• 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.
• 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
• the disclosure provides a hybridoma producing the antigen- binding protein (e.g. anti-MrkA antibodies or antigen binding fragments thereof) of the disclosure.
• a yet further aspect of the disclosure provides a method of production of an
• Such a method can comprise culturing host cells under conditions suitable for production of said antigen-binding protein, MrkA polypeptide, or immunogenic fragment thereof.
• the method of production further comprises isolating
• antigen binding protein including antibodies or antigen binding fragments thereof, MrkA polypeptide, or immunogenic fragment thereof produced from the host cell or hybridoma.
• Klebsiella infections a novel functionally-based screening assay was used to identify cross- protective targets for the Gram negative bacterium K. pneumoniae.
• This novel assay identified antibodies capable of inducing opsonophagocytic killing (OPK) and did not focus, at the outset, on any particular target antigen.
• OPK opsonophagocytic killing
• pneumoniae 43816 strain (43816AcpsBAWaaL or 43816DM) was constructed through allelic replacement with plasmids containing CpsB and WaaL ORFs and selected in the presence of gentamicin. Gentamicin resistant colonies were picked and expanded. The deletions of the CpsB and WaaL genes were confirmed by PCR analysis.
• K. pneumoniae strains expressing luciferase Lux strain
• various K. pneumoniae clinical isolates were transformed with a plasmid containing the luciferase reporter gene and gentamicin resistant colonies were selected. Unless stated otherwise, all K. pneumoniae cultures were maintained in 2xYT media at 37°C, supplemented with antibiotics when appropriate. Phage panning and screening
• 9xl0 9 K. pneumoniae cells from 43816AcpsBAWaaL were used as the panning antigen for round one, followed by two more rounds of panning on an equal mix of wild type strains 1901 (ATCC BAA-1901) and 1899 (ATCC BAA-1899).
• wild type strains 1901 ATCC BAA-1901
• 1899 ATCC BAA-1899
• bacterial cells were harvested at mid-log phase and blocked (2xYT + 3 % dry milk), followed by addition of lxlO 12 blocked phage particles. Cells were then washed seven times by repeated re-suspension in PBS. Bound phage particles were eluted with 0.
• mice were immunized with 43816AcpsBAWaaL via intraperitoneal (LP.) route weekly for four weeks followed by a final boost with a mixture of wild type K.
• LP. intraperitoneal
• lymph node lymphocytes and splenocytes were harvested and fused with P3X myelomas and subjected to selection in lx HAT culture medium. Supernatants from the resulting hybridomas were then screened for binding to 43816AcpsBAWaaL by whole bacterial ELISA. Positive binders were subjected to the high-throughput OPK assay to select for potentially protective hybridomas against K. pneumoniae.
• ELISA as described in DiGiandomenico, et al., J Exp Med, 209: 1273-87 (2012), herein incorporated by reference. Briefly, a single colony of K. pneumoniae was inoculated into 2xYT media until the culture reached log phase. Bacteria were coated onto 384-well plates (Nunc MaxiSorp) overnight at 4°C. A set of plates were coated with similarly prepared culture of Acinetobactor pitti 19004 (ATCC 19004) as negative controls. After blocking with PBS supplemented with 4% BSA (PBS-B), the coated plates were incubated with anti- . pneumoniae antibodies for lh.
• OPK assays were performed based on the procedure described in
• K. pneumoniae 43816 was grown overnight in 2xYT culture medium at 37°C.
• Fluorescent labeling was achieved by incubating bacteria with the MrkA specific monoclonal antibody Kp3, followed by Alexa 488 labeled anti-human IgG secondary antibody
• K. pneumoniae overnight culture was collected by centrifugation, and the cell pellet was re-suspended in 3 ml of B-PER (Thermo Scientific) buffer supplemented with protease inhibitor cocktail and DNasel (2 ⁇ /ml at 200 U/ ⁇ ). After incubating at room temperature for 40 min, the supernatant was collected through centrifugation at top speed in a table top Eppendorf centrifuge (14,000 rpm/min) for 20 min at 4°C. The cleared lysate was mixed with 40 ⁇ of protein A/G beads (Pierce, # 20422) and incubated at 4°C for 2 hours.
• B-PER Thermo Scientific
• the lysate was collected by centrifugation again at top speed (14,000 rpm/min) for 15 min at 4°C.
• the cleared lysate was moved to a new Eppendorf tube containing 15 ⁇ of protein A/G beads (prewashed with B-PER), 6 ⁇ g of immunoprecipitation antibody, incubated on a rotator for 3 hours at 4°C.
• the beads were then collected by spinning at 10,000 rpm, 1 min at 4°C followed by three washes with ice cold B-PER buffer.
• Immunoprecipated samples were then re-suspended in SDS-PAGE buffer and loaded directly onto a SDS-PAGE gel (4-12% gradient gel Novex). Half of the sample was loaded on one gel for blue stain (Invitrogen) and subsequent mass spec sample preparation; the other half was loaded to a second gel for Western blot analysis.
• MrkA-his tag open reading frame (ORFs) was synthesized, cloned into the expression vector p AC YC-duet-1 (EMD Millipore), and transformed into E. coli BL21 (DE3) cells. Chloramphenicol-resistant colonies were picked and expanded in LB media containing 150 ⁇ g/ml of chloramphenicol. Once the OD (600 nm) reached 0.4, 1 mM IPTG was added to the culture to induce the expression of MrkA-his at 37°C for 4 hours. Bacteria were lysed with B-PER, and the presence of MrkA-his was examined by Western blot using anti-his or MrkA specific mAbs as described herein.
• ORFs The MrkA-his tag open reading frame
• the DNA templates of MrkA for in vitro expression were amplified by PCR.
• the template includes a T7 promoter at the 5', a c-Myc tag and T7 terminator at 3' of MrkA ORF.
• 250 ng of DNA templates were added to the PURExpress in vitro protein system (NEB E6800) with or without Disulfide Bond Enhancer (NEB E6820S) in 25 ⁇ of reaction mixture, and the reaction mixes were incubated at 37°C for 2 hours.
• the synthesized proteins were analyzed by western blot using anti-c-Myc and MrkA specific mAb as described herein.
• C56/BL6 mice were received from Jackson laboratories and maintained in a
• the lung bacterial burden was measured by plating lung homogenates onto agar plates to determine CFU 24 hours post infection.
• C57/bl6 mice were inoculated intranasally with 5e3 CFU or le8 CFU of K. pneumoniae 43816 strain (01 :K2) or K. pneumoniae 985048 strain, respectively.
• Kp3 and human IgGl control antibody were given one day prior to bacterial challenge.
• Mouse survival was monitored daily until up to day 8. Combined survival data of three experiments were plotted in Prism.
• Kp3 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.
• (43816AcpsBAWaaL) was generated to drive the selection process toward more conserved surface antigens.
• the first round of affinity selective panning was performed on 43816DM, followed by two more rounds of panning on a mixture of wild-type isolates (1901 and 1899). More than a hundred-fold enrichment was observed from output titers over three rounds of panning.
• the phage libraries used in this study were single chain fragment variable (scFv) libraries.
• scFv single chain fragment variable
• OPK organic kinase
• the third round panning output was batch-converted into scFv-Fc format.
• This platform allows for scFv-Fc expression in both bacterial and mammalian hosts, which is suitable for both high throughput and functional screening needs.
• the scFV-Fc clones were expressed in bacteria, and the resulting supematants were tested for binding to three live K. pneumoniae wild type strains.
• Kp3 and Kpl6 also showed OPK activities against a panel of K. pneumoniae of different serotypes (Figure IF). Further testing with an expanded spectrum of seven hundred recent K. pneumoniae clinical isolates, Kp3 bound to more than 62% of the strains, with majority of them being multi-drug resistant isolates. A list of representative K. pneumoniae clinical isolates recognized by Kp3 is shown in Table 5.
• 43816DM (43816AcpsBAWaaL) strain was used to immunize mice with the goal to elicit antibodies against antigens different from capsule or LPS O-antigen. After the initial phase of immunization with mutant strain, a final boost was performed with a combination of wild-type strains (1901 and 1899) before spleens and lymph nodes were collected for hybridoma generation. Whole-cell bacterial screening by binding was initially applied in hybridoma generation similarly as the above phage panning approach to identify cross- reactive antibodies. Of the approximately 9000 hybridomas tested, four hybridomas (21G10, 22B12, 88D10, and 89E10) showed serotype independent binding to the K. pneumoniae strains tested ( Figures 1 A and E).
• Kp3 and 88D10 captured four major protein bands with band 1 from a negative control isolate 1899 (Figure 2B). Among them, band number 3 is reactive to Kp3 in a Western blot analysis ( Figure 2C). All four bands were excised and subjected to LC-MS analysis. The most dominant protein band (Figure 2B band #3) was identified as MrkA, as peptides covering more than 50% of the full MrkA sequence were recovered. MrkA peptides identified through mass spectrometry are shown in bold face and underlined in Figure 2D.
• the other dominant band (Figure 2B band #2) was identified as MrkB, a chaperon protein that facilitates MrkA fimbrial subunit folding and transportation through the periplasmic space. (Chan et al., Langmuir 25:7428-35 (2012); Burmolle et al., Microbiology 154: 187-95 (2008).)
• the least dominant band (Figure 2B band #4) and one isolated from the negative control isolate (Figure 2B band #1) did not identify any specific cell surface localized protein.
• MrkA was the single protein species identified from Figure 2B band No.
• MrkA ORF of strain MGH78578 from the UniProt database was cloned into an expression vector and expressed in BL21 cells. Lysates were then prepared using B-PER and subjected to Western blot analysis using an anti-his tag or Kp3. Similar to the endogenous MrkA, the recombinant MrkA displayed a laddered pattern including bands ranging in apparent sizes from 60 kDa to more than 200kDa ( Figure 3 A). Interestingly, while the anti-his antibody recognized both monomeric and oligomeric MrkA, Kp3 recognized only the oligomeric form. The MrkA mAb target identity is also consistent with the fimbriae structure shown in confocal experiments ( Figure 2A).
• MrkA Recombinant MrkA was also expressed with a c-Myc tag in an in vitro transcription and translation system under different experiment conditions, and the products were subjected to Western blot analysis.
• in vitro expression system predominantly produced MrkA monomeric protein (Figure 3B).
• Kp3 recognized higher molecular weight bands present in bacterial cell lysate ( Figure 3B, sample 1), it was not able to detect the MrkA monomer. This suggests that Kp3 binds to high order MrkA structures in type III fimbriae and that the MrkA assembly may require the contribution of other cellular components or conditions which are lacking in the in vitro expression system used in this study.
• Kp3 was evaluated in a murine model of K.
• Kp3 was also tested in a lethal pneumonia model using Kp43816, a virulent
• MrkA-N-dlt N-terminal deletion
• MrkA-C-dlt 32 amino acid C-terminal deletion
• MrkA-N/C-dlt combination of the N- and C-terminal deletions
• Bacterial cells were then collected and subjected to lysis using B-PER Bacterial Protein Extraction Reagent (Thermo Scientific). The clear cell lysate was used directly to coat an ELISA plate, and binding of Kp3 was measured using a standard ELISA procedure. Human IgGl and an unrelated anti-MrkA antibody were used as controls.
• Kp3 only detected full length MrkA and did not bind to: MrkA-N-dlt; i.e., amino acids 41-202 of SEQ ID NO: 17 (i.e., SEQ ID NO:26); MrkA-C-dlt; i.e., amino acids 1-170 of SEQ ID NO: 17 (i.e., SEQ ID NO:27) or MrkA-N/C-dlt; i.e., amino acids 41-170 of SEQ ID NO: 17 (i.e., SEQ ID NO:28).
• a control anti-MrkA antibody detected full length MrkA as well as MrkA with N terminal deletion (data not shown).
• Example 8 Monomeric and polymeric MrkA reduce organ burden in a bacterial challenge model
• MrkA Recombinant MrkA protein exists in both monomeric and polymeric form ( Figure 3 A).
• Figure 3 A In order to assess the role of monomeric and oligomeric MrkA protein in inducing protective immunity, both monomeric and polymeric species were purified by column fractionations. Briefly, in order to express MrkA on a large scale, the mature form of MrkA (SEQ ID NO: 17) was cloned into pET28 (Novagen) in frame with an N terminal 6 X his tag. The protein was expressed by the host BL21-DE3 E. coli strain.
• Transformed cells were grown in Terrific Broth (Corning) + Kanamycin (50 ⁇ g/ml) at 37°C with 250 RPM shaking until reaching an OD600 of 0.6.
• IPTG (1 M) (InVitrogen) was added to the culture for a final concentration of 1 mM, and the culture was incubated for an additional 4 hours.
• the cells were harvested by centrifugation (12,000 X g for 10 minutes), and the cell pellet was stored at -80°C until purification.
• solubilization buffer 10 mM Tris, pH 8, 100 mM sodium phosphate, 8 M Urea, 1 mM DTT. Solubilized inclusion bodies were clarified by centrifugation at 27,000 x g for 15 minutes at 10°C then loaded onto a 5 ml HisTrap HP column (GE Healthcare) equilibrated with solubilization buffer. Both flow through and eluted fractions were collected and subjected to refolding according to the described protocol.
• Refolded mixtures were loaded onto a HisTrap column and eluted with a linear gradient up to 500 mM Imidazole in 25 mM sodium phosphate, pH 7.4 with 500 mM NaCl.
• Monomeric MrkA was collected early in the gradient (approx. 150 mM Imidazole) and oligomeric species later in the gradient (approx. 250 mM Imidazole).
• Each pool was concentrated with Vivaspin 5 K MWCO devices (Vivascience) and dialyzed into 10 mM Tris, pH 7.5 with 100 mM NaCl.
• Buffer [10 mM Tris, 100 mM sodium phosphate, 1 mM EDTA, 5 mM Cysteamine, 0.5 mM Cystamine, pH 8]. They were allowed to mix overnight at 4°C. They were dialyzed into refolding buffer (Dilution Buffer without EDTA) at 4°C (two exchanges) then into IX PBS, pH 7.2. The dialyzed samples were purified using HisTrap (eluted with a linear gradient to 500 mM Imidazole in 25 mM sodium phosphate, pH 7.4 with 500 mM NaCl).
• MrkA that was retained in the column during the first loading step contained mostly oligomeric MrkA. It was refolded on the column, eluted, and concentrated as described above. Purification from inclusion body resulted in monomeric and oligomeric MrkA with high purity ( Figure 7), which was used in subsequent immunization experiments.
• biofilm assays were performed according to Wilksch et al., (PLos Pathogens 7(8): el002204 (2011)) with modifications.
• K. pneumoniae 43816 were allowed to grow into log phase culture and diluted into minimum media (RPMI-1%BSA) to be OD 650 equals to 0.1.
• RPMI-1%BSA minimum media
• bacteria were incubated in flat bottom, 96 well microtiter plates (Falcon; BD Biosciences) with a series dilution of Kp3 or hlgGl (isotype control) antibodies.
• Type III fimbriae in K. pneumoniae are filamentous appendages that mediate adherence to eukaryotic cells and abiotic surfaces. MrkA, a major fimbrial subunit, but not adhesin (MrkD) were previously shown to facilitate biofilm formation (Langstraat et al., Infect Immun 2001; 69:5805-12).
• Kp3 or hlgGl (isotype control) antibodies were added to confluent A549 cells grown in opaque 96-well plates (Nunc Nunclon Delta). Log-phase luminescent K pneumoniae 43816 was added at a multiplicity of infection (MOI) of 50. After incubation at 37°C for 90 min, cells were washed, followed by the addition of 0.05 ml of 2xYT + 0.5% glucose.
• a target agnostic strategy was applied to identify cross-protective antibodies for the treatment of K. pneumoniae infection. While significant efforts have been made to identify cross-reactive antibodies targeting K pneumoniae, there are major obstacles in developing such therapeutics.
• Well validated antibody targets including CPS and LPS are serotype specific and therefore require multiple antibodies for broad strain coverage. This challenge was overcome by constructing CPS and LPS O-antigen deletion mutants to focus on more conserved surface antigens.
• anti-MrkA antibodies from both hybridoma and phage display platforms demonstrating significant in vitro and in vivo efficacies against Klebsiella were identified.
• MrkA is a major protein of the type III fimbriae complex and has been implicated in host cell attachment and biofilm formation (see Murphy et al, Future Microbiol 2012; 7:991-1002), a strategy bacterial pathogens use to establish infection (Burmolle et al., Microbiology 2008; 154: 187-95).
• mice immunized with purified type III fimbriae displayed resistance to subsequent ⁇ , pneumoniae challenge, albeit only to relatively low challenging doses (Lavender et al., International journal of medical microbiology 2005; 295: 153-9). Although humoral immunity was implicated as the protective mechanism, the antigenic components that elicited protection were not elucidated.
• anti-MrkA monoclonal antibodies disclosed herein contribute to the immune protection through multiple mechanisms.
• antibodies against type III fimbrial adhesin protein MrkD showed cross-reactivity to multiple K. pneumoniae strains similar to anti-MrkA mAbs, but did not induce OPK and confer no protection in vivo (data not shown). This further confirmed that OPK activity may be necessary for in vivo protection for these antibodies.
• MrkA A promising feature of MrkA as an antibody therapeutic target is its high degree of sequence conservation among different isolates and general accessibility as an
• MrkA from the two most dominant pathogenic isolates K. pneumoniae and K. oxytoca have a 95% homology, and the homologies among representative members of the Enterobacterecea family are more than 90% with the exception of Enterobacter cloacae, which is divergent from the rest ( Figure 5). Further work is needed to survey extensively the MrkA sequences from other members. Nevertheless this presents a potential opportunity to develop a MrkA-based anti- . pneumoniae and pan Gram negative strategy.
• MrkA his-tagged recombinant MrkA was expressed and purified as described in the materials and methods section with modifications. MrkA expressed in the E. coli host strain BL21(DE3) stayed mostly in the inclusion body. Buffer containing eight molar urea was used to solubilize MrkA, and the his-tagged MrkA was purified using HisTrap HP column (GE Healthcare) as described previously (see Wang, Q. et al, 2016. Target Agnostic Identification of Functional Monoclonal Antibodies against Klebsiella pneumoniae Multimeric MrkA Fimbrial Subunit.
• MrkA MrkA
• the panning output was improved more than 100-fold compared to the first round.
• the panning output was converted to scFv.Fc in pSplice.V5 and subjected to high throughput screening as described above and summarized in Figure 11, with further illustration of the homogeneous time resolved FRET (HTRF) process in Figure 12.
• HTRF time resolved FRET
• the four clones 1, 4, 5, and 6 were expressed as human IgGl in 293 free style cells (Invitrogen) and purified. While they maintained robust binding activities, the ELISA format impacted the bindings significantly. Their apparent affinities are between 3-10 nM ( Figure 13 and Table 7) as measured by BLI approach in an IgG format. Western blot data showed that only clone 1 was able to detect the monomeric MrkA, whereas none of the others were able to do so ( Figure 14).
• MrkA is especially intolerant to mutations, and sub-clones and expression of fragments from MrkA often resulted in no expression.
• mutational analysis is not a suitable method for epitope analysis. Instead, the BLI-based approach for studying the relative positions of the epitopes of the mAbs was used. Epitope binning was done on a ForteBio Octet QK384. Biotinylated-MrkA was captured onto streptavidin biosensors and coated with testing mAbs at a saturating concentration of 200 nM for 600 seconds.
• Table 8 K D measurement in Fab format against monomeric MrkA. ND, not detectable; N/A, not applicable.
• clone 1 binds to a non-overlapping epitope of MrkA as well as to monomeric MrkA.
• Example 12 OPK activity is important for in vivo protection
• Example 13 Antibody binding to live bacteria as exemplified by flow cytometry
• RLUs relative light units
• the anti-MrkA antibodies did show protective activity in vivo.
• Example 16 Single antibodies are as protective as antibody combinations

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