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WO2013078223A1 - Anticorps anti-protéine liant le facteur h et procédés pour les utiliser - Google Patents

Anticorps anti-protéine liant le facteur h et procédés pour les utiliser Download PDF

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WO2013078223A1
WO2013078223A1 PCT/US2012/066099 US2012066099W WO2013078223A1 WO 2013078223 A1 WO2013078223 A1 WO 2013078223A1 US 2012066099 W US2012066099 W US 2012066099W WO 2013078223 A1 WO2013078223 A1 WO 2013078223A1
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Prior art keywords
antibody
fhbp
jar
seq
amino acid
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Dan M. Granoff
David Manh-Tung VU
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Childrens Hospital Oakland Research Center
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Childrens Hospital Oakland Research Center
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    • 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
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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

Definitions

  • Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis.
  • fHbp Factor H Binding Protein
  • LP2086 Genome-derived Neisserial antigen (GNA) 1870, or “741”
  • fHbp human complement factor H
  • Factor H binding proteins can be subdivided into two sub-families (A and B); three variant groups (Groups 1, 2, and 3); or ten modular groups (Groups TX), based on amino acid sequence identity. Six of the ten modular groups account for 99% of all known sequence variants.
  • the present disclosure provides antibodies that bind specifically to factor H binding protein.
  • the antibodies are useful in various applications, which are also provided.
  • a subject isolated antibody competes for binding with an antibody that comprises: (i) a V L CDRI comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 14; (ii) a V L CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ ID NO: 15; (iii) a V L CDR3 comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO: 16; (iv) a V H CDRI comprising an amino acid sequence of SEQ ID NO:l or SEQ ID NO: 11; (v) a V R CDRI comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 12; and (vi) a V H CDR3 comprising an amino acid sequence of SEQ ID NO:3 or SEQ
  • An isolated antibody of the present disclosure binds the epitope (an epitope of a fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3) with an affinity of from about 10 7' M 1 to about 101 1 2" M 1.
  • the heavy chain region of a subject anti-fHbp antibody is of the isotype IgGl, IgG2, IgG3, or IgG4.
  • a subject anti-fHbp antibody is a Fv, scFv, Fab, F(ab')2, or Fab'.
  • a subject anti-fHbp antibody is a single chain Fv (scFv) antibody, e.g., a multimerized scFv. In some cases, a subject anti-fHbp antibody is a humanized antibody.
  • scFv single chain Fv
  • a subject anti-fHbp antibody is a humanized antibody.
  • Any of the aforementioned anti-fHbp antibodies of the present disclosure can comprise a detectable label. Any of the aforementioned anti-fHbp antibodies of the present disclosure can be immobilized on a solid support. Any of the aforementioned anti-fHbp antibodies of the present disclosure can comprise a polyamine modification.
  • a subject anti-fHbp antibody competes for binding with an antibody produced by the hybridoma deposited as ATCC Deposit No. PTA- 12202 and/or the hybridoma deposited as ATCC Deposit No. PTA- 12201.
  • a subject anti-fHbp antibody is the antibody produced by the hybridoma deposited as ATCC Deposit No PTA- 12202.
  • a subject anti-fHbp antibody is the antibody produced by the hybridoma deposited as ATCC Deposit No. PTA- 12201.
  • the present disclosure provides an isolated nucleic acid comprising a nucleotide sequence encoding a subject isolated antibody that specifically binds an epitope in a fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3.
  • the present disclosure provides a recombinant expression vector comprising a subject nucleic acid, where the nucleotide sequence is operably linked to a transcriptional control element that is active in a prokaryotic or a eukaryotic cell.
  • the present disclosure also provides in vitro host cell genetically modified with an isolated nucleic acid comprising a nucleotide sequence encoding a subject isolated antibody that specifically binds an epitope in an fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3.
  • the present disclosure also provides in vitro host cell genetically modified with a recombinant expression vector comprising a subject nucleic acid, where the nucleotide sequence is operably linked to a transcriptional control element that is active in a prokaryotic or a eukaryotic cell.
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising: a) a subject isolated antibody that specifically binds an epitope in an fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3; and b) a pharmaceutically acceptable carrier.
  • the present disclosure provides a method of detecting a factor H binding protein
  • the method generally involves: (a) contacting the test sample with a subject isolated antibody that specifically binds an epitope in an fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3; and (b) detecting binding, if any, of the antibody to an epitope present in the test sample. Binding of the antibody indicates that the test sample comprises an fHbp.
  • the antibody is immobilized on a solid support.
  • the antibody comprises a detectable label.
  • the fHbp being detected can be present on the surface of a bacterium. Alternatively, the fHbp being detected can be in solution.
  • the present disclosure provides a method of treating a Neisseria meningitidis infection in an individual, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising: a) a subject isolated antibody that specifically binds an epitope in an fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3; and b) a pharmaceutically acceptable carrier.
  • the treatment method can also comprise administering a second antibody specific for an fHbp. In some cases, the combination of the antibody with the second antibody provides for bactericidal activity toward at least one N.
  • Figure 1 provides the amino acid sequence of human factor H (fH), which is also provided in GenBank accession no. NP_000177 (P08603); the nucleotide sequence encoding this fH polypeptide is provided in GenBank Accession No. NM_000186.
  • fH human factor H
  • Figure 2 provides an alignment of amino acid sequences of natural fHbp variants.
  • Figure 3 depicts concentration-dependent binding of JAR 41 monoclonal antibody
  • Figure 4 depicts concentration-dependent binding of JAR 64 mAb to the nineteen different fHbp variants.
  • Figure 5A provides a network analysis of amino acid sequences of fHbp variants recognized by JAR 41 and JAR 64 mAbs. See also Pajon et al. (2010) Vaccine 28:2122.
  • fHbp sequence variants of known amino acid sequence. See, e.g., the web site pubmlst(dot)org/Neisseria/fHbp/.
  • fHbp variants that are representative of the known fHbp sequences depicted schematically in Figure 5 were analyzed for binding by JAR 64 or JAR 4 antibodies.
  • the 19 fHbp variants tested are representative of the sub-families (A and B), variant groups (v.l, v.2, and v.3) and modular groups (I-X) to which all known fHbp variants belong.
  • Figure 5B provides a network analysis of fHbp variants recognized by JAR 4
  • Figure 6 (left panel) illustrates the inhibition by ELISA of binding of alkaline phosphatase-conjugated JAR 41 to fHbp ID 1 by JAR 64 or JAR 4.
  • the right panel shows the inhibition of binding of unconjugated JAR 4 (IgG2a) to fHbp by JAR 41 (IgGl) or JAR 64 (IgGl).
  • Figure 7 depicts the binding of anti-fHbp mAb JAR 41 (Panel A) and JAR 64
  • Figure 8 illustrates the cooperative bactericidal activity of JAR 41 against strain
  • Figure 9 illustrates the cooperative bactericidal activity of JAR 41 against strain
  • Figures 10A and 10B provide JAR 41 heavy and light chain variable region nucleotide and amino acid sequences.
  • Figures 11 A and 1 IB provide JAR 64 heavy and light chain variable region nucleotide and amino acid sequences.
  • Figure 12 shows the amino acid alignment of the complementarity determining regions (CDRs) of both JAR 41 and of JAR 64 aligned with the respective germline CDR amino acid sequences. The corresponding alignment for a third anti-fHbp mAb, JAR 4, is shown for comparison.
  • JAR 41 H chain CDRs CDR1: NYYIY (SEQ ID NO: l);
  • CDR2 CDR1: NYYIY (SEQ ID NO: l);
  • JAR41 L chain CDRs CDR1: KASQSVDYDGKSYMN (SEQ ID NO:4); CDR2: VASNLES (SEQ ID NO:5); and CDR3: QQSNEDPWT (SEQ ID NO:6).
  • JAR64 H chain CDRs CDR1: DYYMH (SEQ ID NO: 11); CDR2: RINPYNGATSYNQNFKD (SEQ ID NO: 12); CDR3: DIYSRWFAY (SEQ ID NO: 13).
  • JAR64 L chain CDRs CDR1: KASQSVDYDGNSYMN (SEQ ID NO: 14); CDR2: VASILES (SEQ ID NO: 15); and CDR3: QQSNEDPWS (SEQ ID NO: 16).
  • JAR4 H chain CDRs CDR1: DYYMN (SEQ ID NO:41); CDR2: RVNPS NG ATTYNQKFKG (SEQ ID NO:42); CDR3: DWEGPWFAY (SEQ ID NO:43).
  • KSSQTLFYSKGKTYLN SEQ ID NO:44
  • CDR2 LVSKLDS (SEQ ID NO:45)
  • CDR3 LQSTHFPYT (SEQ ID NO:46).
  • Figure 13 depicts binding of JAR 3 and JAR 41 to wildtype or mutant fHbp expressed by yeast cells as measured by flow cytometry.
  • Figure 14 depicts binding by ELISA of JAR 41, JAR 64, JAR 4 and JAR 5 to fHbp ID 1 or mutants of fHbp ID 1 with single amino acid substitutions.
  • Figure 15 is Table 5 that lists numerous fHbp variants, available in the database at neisseria.org. The variant group, sub-family, and modular group to which each fHbp variant belongs are provided. GenBank Accession numbers for fHbp variants are also provided. As noted above, amino acid sequences of about 553 fHbp variants are available. See, e.g., the web site pubmlst(dot)org/Neisseria/fHbp/. Figure 15 lists a fraction of the known fHbp variants.
  • Figure 16 depicts passive protective activity of JAR 41 and JAR 5 in human fH transgenic infant rats.
  • Human factor H refers to a protein comprising an amino acid sequence as shown in Figure 1 (SEQ ID NO:21), and naturally- occurring human allelic variants thereof.
  • Fractor H Binding Protein (fHbp), which is also known in the literature as
  • GNA1870, ORF2086, LP2086 (lipoprotein 2086), and "741" refers to a class of N. meningitidis polypeptides. It is found in nature as a lipoprotein on the surface of the bacterium N.
  • fHbps meningitidis.
  • fHbps have been sub-divided into three fHbp variant groups (referred to as variant 1 (v.l), variant 2 (v.2), and variant 3 (v.3) in some reports (Masignani et al. (2003) J Exp Med 197:789-99), and Family A and B in other reports (see, e.g., Fletcher et al. (2004) Infect. Immun. 72:2088-2100) based on amino acid sequence variability and immunologic cross -reactivity (Masignani et al. (2003) J Exp Med 197:789-99).
  • ID fHbp peptide identification
  • the fHbp amino acid sequence variants also can be assigned into modular groups based on six variable segments, each derived from one of two genetic lineages. Beernink and Granoff (2009) Microbiol. 155:2873; and WO 2010/127172.
  • variant 2 (v.2) fHbp protein (from strain 8047, fHbp ID 77) and variant 3 (v.3) fHbp (from strain M1239, fHbp ID 28) differ by -1 and +7 amino acid residues, respectively, from that of MC58 (fHbp ID 1)
  • the numbering used to refer to residues for v.2 and v.3 fHbp proteins differs from numbering based on the actual amino acid sequences of these proteins.
  • reference to a leucine residue (L) at position 166 of the v.2 or v.3 fHBP sequence refers to the residue at position 165 of the v.2 protein and at position 173 in the v.3 protein.
  • Examples of fHbp are shown in Figure 2.
  • Serogroup refers to classification of Neisseria meningitidis by virtue of immunologically detectable variations in the capsular polysaccharide. About 12 serogroups are known: A, B, C, X, Y, Z, 29-E, W-135, H, I, K and L. Any one serogroup can encompass multiple serotypes and multiple serosubtypes.
  • Serotype refers to classification of Neisseria meningitidis strains based on monoclonal antibody defined antigenic differences in the outer membrane protein Porin B (PorB). A single serotype can be found in multiple serogroups and multiple serosubtypes.
  • Sero subtype refers classification of Neisseria meningitidis strains based on antibody defined antigenic variations on an outer membrane protein called Porin A (PorA), or upon VR typing of amino acid sequences deduced from DNA sequencing (Sacchi et al., 2000, J. Infect. Dis. 182: 1169; see also the Multi Locus Sequence Typing web site).
  • PorA proteins Most variability between PorA proteins occurs in two (loops I and IV) of eight putative, surface exposed loops.
  • the variable loops I and IV have been designated VR1 and VR2, respectively.
  • a single serosubtype can be found in multiple serogroups and multiple serotypes.
  • antibodies and "immunoglobulin” include antibodies or
  • immunoglobulins of any isotype fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fab', F(ab') 2 , Fv, scFv, and Fd fragments; chimeric antibodies; humanized antibodies; single-chain antibodies; and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin-avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the term are Fab', Fv, F(ab') 2 , and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies.
  • An antibody may be monovalent or bivalent.
  • Antibody fragments comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen- binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, a designation reflecting the ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • Fv is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRS of each variable domain interact to define an antigen-binding site on the surface of the V H -V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the "Fab” fragment also contains the constant domain of the light chain and the first constant domain (CH of the heavy chain.
  • Fab fragments differ from Fab' fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHi domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • immunoglobulins The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgA, and IgA2.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, I
  • Single-chain Fv or “sFv” antibody fragments comprise the V H and V L domains of antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the V H and V L domains, which enables the sFv to form the desired structure for antigen binding.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (V H ) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -V L ).
  • V H heavy-chain variable domain
  • V L light-chain variable domain
  • the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
  • Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
  • affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as a dissociation constant (Kd).
  • Kd dissociation constant
  • Affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40- fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80- fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more, than the affinity of an antibody for unrelated amino acid sequences.
  • Affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100
  • fM femtomolar
  • vidity refers to the resistance of a complex of two or more agents to dissociation after dilution.
  • immunosorbent and “preferentially binds” are used interchangeably herein with respect to antibodies and/or antigen- binding fragments.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and/or hydrogen-bond interactions, including interactions such as salt bridges and water bridges.
  • Non-specific binding would refer to binding with an affinity of less than about 10 "7 M, e.g., binding with an affinity of 10 "6 M, 10 "5 M, 10 "4 M, etc.
  • CDR complementarity determining region
  • CDR complementarity determining region
  • MacCallum references are set forth below in Table 1 as a comparison.
  • Residue numbering follows the nomenclature of Kabat et al., supra Residue numbering follows the nomenclature of Chothia et al., supra Residue numbering follows the nomenclature of MacCallum et al., supra
  • variable region when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody.
  • a variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs.
  • framework region is intended to mean each domain of the framework that is separated by the CDRs.
  • An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified (1) to greater than 90%, greater than 95%, or greater than 98%, by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N- terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or nonreducing conditions using Coomassie blue or silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. In some instances, isolated antibody will be prepared by at least one purification step.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • a disease caused by a strain of Neisseria meningitidis encompasses any clinical symptom or combination of clinical symptoms that are present in an infection of a human with a Neisseria meningitidis. These symptoms include but are not limited to:
  • colonization of the upper respiratory tract e.g. mucosa of the nasopharynx and tonsils
  • a pathogenic strain of Neisseria meningitidis penetration of the bacteria into the mucosa and the submucosal vascular bed, septicemia, septic shock, inflammation, haemorrhagic skin lesions, activation of fibrinolysis and of blood coagulation, organ dysfunction such as kidney, lung, and cardiac failure, adrenal hemorrhaging and muscular infarction, capillary leakage, edema, peripheral limb ischaemia, respiratory distress syndrome, pericarditis and meningitis.
  • the present disclosure provides isolated antibodies that bind specifically to factor
  • H binding protein The antibodies are useful in various applications, which are also provided.
  • An anti-fHbp antibody of the present disclosure specifically binds a factor H binding protein (fHbp).
  • An epitope of a fHbp can be formed by a polypeptide having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to a contiguous stretch of from about 50 amino acids (aa) to about 100 aa, from about 100 aa to about 200 aa, from about 200 aa to about 250 aa, or from about 250 aa to about 265 aa, of an amino acid sequence depicted in Figure 2 (SEQ ID NOs:22-40).
  • a subject anti-fHbp antibody binds fHbp from a wide variety of N. meningitidis strains, e.g., strains of N. meningitidis of a variety of serogroups, serotypes, or serosubtypes.
  • an isolated anti-fHbp antibody of the present disclosure specifically binds an epitope in fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3. Masignani et al. (2003) J. Exp. Med. 197:789 or modular groups I to X (Beernink et al (2009) Microbiology 155:2873; and Pajon et al. (2010) Vaccine 28:2122).
  • a subject anti-fHbp antibody binds an epitope that is present in an fHbp that is present in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, fHbp that are in variant group 1. In some cases, a subject anti-fHbp antibody binds an epitope that is present in an fHbp that is present in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, fHbp that are in variant group 2.
  • a subject anti-fHbp antibody binds an epitope that is present in an fHbp that is present in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, fHbp that are in variant group 3.
  • a subject anti-fHbp antibody binds a recombinant fHbp of any of modular groups I-X.
  • an isolated anti-fHbp antibody of the present disclosure specifically binds an epitope in fHbp, which epitope is present in an fHbp of modular groups I-X
  • For modular groups I-X see, e.g., Pajon et al. (2010) Vaccine 28:2122; and Figure 5 (Example 1).
  • a subject anti-fHbp antibody binds fHbp ID 1, ID 4, ID 6, ID 9, ID
  • Variant group 1 fHbp include ID 1, ID 4, ID 6, ID 9, ID 13, ID 14, ID 74, ID 15, and ID 55.
  • Variant group 2 fHbp include ID 19, ID 22, and ID 77.
  • Variant group 3 fHbp include ID 28, ID 45, ID 67, ID 79, and ID 175. Two natural hybrids are designated ID 207 (variant2/l) and ID 283 (variant 3/1).
  • a subject anti-fHbp antibody binds an fHbp of subfamily A and a fHbp of subfamily B.
  • a subject anti-fHbp antibody binds an epitope that is present in an fHbp that is present in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, fHbp that are in subfamily A; and binds an epitope that is present in an fHbp that is present in two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more, fHbp that are in subfamily B.
  • fHbp ID 28, fHbp ID 22, fHbp ID 45, fHbp ID 79, fHbp ID 19, and fHbp ID 77, ID 175, and ID 67 are in subfamily A; while fHbp ID 1, fHbp ID 4, fHbp ID 6, fHbp ID 9, fHbp ID 13, fHbp ID 14, fHbp ID 74, fHbp ID 15, and fHbp ID 55 are in subfamily B.
  • fHbp sequence variants are natural hybrids of sub-Family A and Sub- Family B (for example, fHbp ID 283 and fHbp ID 207) (Illustrated in Figure 5).
  • Figure 2 provides an amino acid sequence alignment of a number of fHbp, including fHbp ID 1, ID 4, ID 6, ID 9, ID 13, ID 14, ID 15, ID 19, ID 22, ID 28, ID 45, ID 55, ID 67, ID 74, ID 77, ID 79, ID 175, ID 207, and ID 283.
  • An anti-fHbp antibody of the present disclosure binds an fHbp variant comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 98%, at least about 99%, or 100%, amino acid sequence identity to the amino acid sequence of an fHbp depicted in Figure 2.
  • a subject antibody exhibits high affinity binding to a fHbp.
  • a subject antibody binds to a fHbp with an affinity of at least about 10 - " 7 M, at least about 10 - “ 8 M, at least about 10 "9 M, at least about 10 "10 M, at least about 10 "11 M, or at least about 10 "12 M, or greater than 10 " M.
  • a subject antibody binds to an epitope present on a fHbp with an affinity of from about 10 "7 M to about 10 ⁇ 8 M, from about 10 ⁇ 8 M to about 10 "9 M, from about 10 "9 M to about 10 "10 M, from about 10 "10 M to about 10 "11 M, or from about 10 "11 M to about 10 "12 M, or greater than 10 "12 M.
  • a subject anti-fHbp antibody competes with a JAR 41 and/or a JAR 64 antibody for binding to a variant group 1 fHbp.
  • a subject anti-fHbp antibody can inhibit binding of a JAR 41 and/or a JAR 64 antibody to a variant group 1 fHbp by more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • a subject anti-fHbp antibody competes with a JAR 41 and/or a JAR
  • a subject anti-fHbp antibody can inhibit binding of a JAR 41 and/or a JAR 64 antibody to fHbp ID 1 by more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • a subject anti-fHbp antibody competes with a JAR 41 and/or JAR 64 antibody for binding to an epitope on an fHbp variant 3 polypeptide, which epitope is not bound by a JAR 4 antibody, and also competes for binding to an epitope present on a variant 1 fHbp polypeptide and a variant 2 fHbp polypeptide.
  • a subject anti-fHbp antibody competes with a JAR 41 and/or JAR 64 antibody for binding to an epitope on an fHbp variant 3 polypeptide (e.g., an fHbp ID 28, ID 45, ID 67, ID 79 or ID 175 polypeptide), which epitope is not bound by a JAR 4 antibody, and also competes for binding to an epitope present on a fHbp variant 1 polypeptide (e.g., an fHbp ID 1, ID 4, ID 6, ID 9, ID 13, ID 14, ID 15, ID 55, or ID 74 polypeptide) and an fHbp variant 2 polypeptide (e.g., an fHbp ID 22, ID 19, or ID 77 polypeptide)
  • an fHbp variant 3 polypeptide e.g., an fHbp ID 28, ID 45, ID 67, ID 79 or ID 175 polypeptide
  • a fHbp variant 1 polypeptide e.g., an
  • a subject anti-fHbp antibody competes with a JAR 41 and/or a JAR 64 antibody for binding to a variant group 2 fHbp.
  • a subject anti-fHbp antibody can inhibit binding of a JAR 41 and/or a JAR 64 antibody to a variant group 2 fHbp by more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • a subject anti-fHbp antibody competes with a JAR 41 and/or a JAR 64 antibody for binding to a variant group 3 fHbp, which epitope is not bound by a JAR 4 antibody.
  • a subject anti-fHbp antibody can inhibit binding of a JAR 41 and/or a JAR 64 antibody to a variant group 3 fHbp by more than 75%, more than 80%, more than 85%, more than 90%, or more than 95%.
  • antibody refers to a protein comprising one or more (e.g., one or two) heavy chain variable regions (VH) and/or one or more (e.g., one or two) light chain variable regions (VL), or subfragments thereof capable of binding an epitope.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions (CDR)", interspersed with regions that are more conserved, termed “framework regions (FR)”.
  • CDR complementarity determining regions
  • FR framework regions
  • a VH can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • a VL can comprise three CDRs and four FRs arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the VH or VL chain of an antibody can further include all or part of a heavy or light chain constant region, to thereby form a heavy or light immunoglobulin chain, respectively.
  • the antibody is a tetramer of two heavy and two light chains, wherein the heavy and light chains are interconnected by, for example, disulfide bonds.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable regions of the heavy and light chains comprise binding regions that interact with antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues and factors, including various cells of the immune system and the first component of the complement system.
  • the term "antibody" includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM and subtypes thereof.
  • a subject antibody is an IgG isotype.
  • immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes; and numerous immunoglobulin variable region genes.
  • Full-length immunoglobulin light chains (about 25 kD or 214 amino acids) are encoded by a variable region gene at the N-terminus (about 110 amino acids) and a kappa or lambda constant region at the C-terminus.
  • Full-length immunoglobulin heavy chains (about 50 kD or 446 amino acids) are encoded by a variable region gene at the N-terminus (about 116 amino acids) and one of the other aforementioned constant region genes at the C-terminus, e.g. gamma (encoding about 330 amino acids).
  • a subject antibody comprises full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain.
  • a subject antibody does not comprise a full-length immunoglobulin heavy chain and a full-length immunoglobulin light chain, and instead comprises antigen-binding fragments of a full-length immunoglobulin heavy chain and a full- length immunoglobulin light chain.
  • the antigen-binding fragments are contained on separate polypeptide chains; in other embodiments, the antigen-binding fragments are contained within a single polypeptide chain.
  • the term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that are capable of specifically binding to fHbp, as described above.
  • binding fragments include (i) a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab') 2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment (consisting of the VH and CHI domains); (iv) a Fv fragment (consisting of the VH and VL domains of a single arm of an antibody); (v) a dAb fragment (consisting of the VH domain); (vi) an isolated CDR; (vii) a single chain Fv (scFv) (consisting of the VH and VL domains of a single arm of an antibody joined by a synthetic linker using recombinant means such that the VH and VL domains pair to form a monovalent molecule); (viii) diabodies
  • a subject antibody fragment is a Fab fragment. In some embodiments, a subject antibody fragment is a single-chain antibody (scFv).
  • a subject antibody is a recombinant or modified antibody, e.g., a chimeric, humanized, deimmunized or an in vitro generated antibody.
  • a recombinant or modified antibody e.g., a chimeric, humanized, deimmunized or an in vitro generated antibody.
  • recombinant or modified antibody as used herein is intended to include all antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (i) antibodies expressed using a recombinant expression vector transfected into a host cell; (ii) antibodies isolated from a recombinant, combinatorial antibody library; (iii) antibodies isolated from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes; or (iv) antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human
  • immunoglobulin gene sequences to other DNA sequences.
  • recombinant antibodies include humanized, CDR grafted, chimeric, deimmunized, and in vitro generated antibodies; and can optionally include constant regions derived from human germline immunoglobulin sequences.
  • a subject antibody specifically binds an epitope in a fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3, and competes for binding to a fHbp with an antibody that comprises:
  • V L CDR1 comprising an amino acid sequence of SEQ ID NO:4 or SEQ ID NO: 14;
  • V L CDR2 comprising an amino acid sequence of SEQ ID NO:5 or SEQ ID NO: 15;
  • V L CDR3 comprising an amino acid sequence of SEQ ID NO:6 or SEQ ID NO: 16;
  • V H CDRI comprising an amino acid sequence of SEQ ID NO:l or SEQ ID NO: 11;
  • V R CDR2 comprising an amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 12;
  • V H CDR3 comprising an amino acid sequence of SEQ ID NO:3 or SEQ ID NO: 13.
  • a subject antibody specifically binds an epitope in a fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3, and competes for binding to a fHbp with an antibody that comprises V L and V H CDRS of JAR 41, e.g., a subject antibody competes for binding to a fHbp polypeptide with an antibody that comprises a V L CDRl comprising SEQ ID NO:4; a V L CDR2 comprising SEQ ID NO:5; a V L CDR3 comprising SEQ ID NO:6; a V H CDRl comprising SEQ ID NO: l, a V H CDR2 comprising SEQ ID NO:2; and a V H CDR3 comprising SEQ ID NO:3.
  • a subject antibody comprises a V L CDRl comprising SEQ
  • V L CDR2 comprising SEQ ID NO:5
  • V L CDR3 comprising SEQ ID NO:6
  • V H CDRl comprising SEQ ID NO: l
  • V H CDR2 comprising SEQ ID NO:2
  • V H CDR3 comprising SEQ ID NO:3.
  • a subject antibody specifically binds an epitope in a fHbp, which epitope is present in an fHbp of variant groups 1, 2, and 3, and competes for binding to a fHbp polypeptide with an antibody that comprises V L and V H CDRS of JAR 64, e.g., a subject antibody competes for binding to a fHbp polypeptide with an antibody that comprises a V L CDR1 comprising SEQ ID NO: 14; a V L CDR2 comprising SEQ ID NO: 15; a V L CDR3 comprising SEQ ID NO: 16; a V H CDR1 comprising SEQ ID NO: 11, a V H CDR2 comprising SEQ ID NO: 12; and a V H CDR3 comprising SEQ ID NO: 13.
  • a subject antibody comprises a V L CDRl comprising SEQ
  • V L CDR2 comprising SEQ ID NO: 15
  • V L CDR3 comprising SEQ ID NO: 16
  • V H CDRl comprising SEQ ID NO: 11
  • V H CDR2 comprising SEQ ID NO: 12
  • V H CDR3 comprising SEQ ID NO: 13.
  • a subject antibody comprises: a variable domain comprising: a) a heavy chain variable domain comprising: i. a CDRl region that is identical in amino acid sequence to the heavy chain CDRl region of the anti-fHbp antibody designated JAR 41; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the JAR 41 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the JAR 41 antibody; and b) a light chain variable domain comprising: i. a CDRl region that is identical in amino acid sequence to the light chain CDRl region of the JAR 41 antibody; ii.
  • a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the JAR 41 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the JAR 41 antibody wherein the antibody specifically binds a fHbp polypeptide.
  • an antibody comprising: a) a variable domain
  • a CDRl region that is identical in amino acid sequence to the heavy chain CDRl region of a JAR 41 antibody; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the JAR 41 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the JAR 41 antibody; and b) a light chain variable domain comprising: i. a CDRl region that is identical in amino acid sequence to the light chain CDRl region of the JAR 41 antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the JAR 41 antibody; and iii.
  • a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the JAR 41 antibody; or b) a variant of the variable domain of part a) that is otherwise identical to the variable domain of part a) except for a number of (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) amino acid substitutions in the CDR regions, where the antibody specifically binds a fHbp polypeptide.
  • a subject anti-fHbp antibody comprises: a) a light chain region comprising: i) one, two, or three complementarity determining regions (CDRs) from the JAR 41 light chain variable region sequence; and ii) a light chain framework region, e.g., a framework region from a human immunoglobulin light chain; and b) a heavy chain region comprising: i) one, two, or three CDRs from the JAR 41 heavy chain variable region sequence; and ii) a heavy chain framework region, e.g., a framework region from a human immunoglobulin heavy chain.
  • CDRs complementarity determining regions
  • a subject anti-fHbp antibody comprises: a variable domain comprising: a) a heavy chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of the anti-fHbp antibody designated JAR 64; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the JAR 64 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the JAR 64antibody; and b) a light chain variable domain comprising: i.
  • a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the JAR 64 antibody; ii. a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the JAR 64 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the JAR 64 antibody, wherein the antibody specifically binds a fHbp polypeptide.
  • a subject anti-fHbp antibody comprises: a) a variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the heavy chain CDR1 region of a JAR 64 antibody; ii. a CDR2 region that is identical in amino acid sequence to the heavy chain CDR2 region of the JAR 64 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the heavy chain CDR3 region of the JAR 64 antibody; and b) a light chain variable domain comprising: i. a CDR1 region that is identical in amino acid sequence to the light chain CDR1 region of the JAR 64 antibody; ii.
  • a CDR2 region that is identical in amino acid sequence to the light chain CDR2 region of the JAR 64 antibody; and iii. a CDR3 region that is identical in amino acid sequence to the light chain CDR3 region of the JAR 64 antibody; or b) a variant of the variable domain of part a) that is otherwise identical to the variable domain of part a) except for a number of (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) amino acid substitutions in the CDR regions, where the antibody specifically binds a fHbp polypeptide.
  • a subject anti-fHbp antibody comprises: a) a light chain region comprising: i) one, two, or three complementarity determining regions (CDRs) from the JAR 64 light chain variable region sequence; and ii) a light chain framework region, e.g., a framework region from a human immunoglobulin light chain; and b) a heavy chain region comprising: i) one, two, or three CDRs from the JAR 64 heavy chain variable region sequence; and ii) a heavy chain framework region, e.g., a framework region from a human immunoglobulin heavy chain.
  • CDRs complementarity determining regions
  • a subject antibody can comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence depicted in Figure 10A and set forth in SEQ ID NO:8.
  • a subject antibody can comprise a heavy chain variable region comprising one, two, or three of the heavy chain complementarity determining regions (CDRs) having an amino acid sequence selected from one or more of SEQ ID NOs: l, 2, and 3.
  • a subject antibody comprises a heavy chain variable region comprising one, two, or three of the heavy chain CDRs having an amino acid sequence selected from one or more of SEQ ID NOs: l, 2, and 3; and FR regions that are human sequences (e.g., encoded by human heavy chain FR-encoding sequences).
  • a subject antibody comprises a heavy chain variable region that comprises, in order from N- terminus to C-terminus: a human heavy chain FR1; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: l; a human heavy chain FR2; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; a human heavy chain FR3; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; and a human heavy chain FR4.
  • a subject antibody can comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence depicted in Figure 10B and set forth in SEQ ID NO: 10.
  • a subject antibody can comprise a light chain variable region comprising one, two, or three of the light chain CDRs having the amino acid sequence set forth in SEQ ID NOs:4, 5, and 6.
  • a subject antibody comprises a light chain variable region comprising one, two, or three of the light chain CDRs having a polypeptide sequence selected from one or more of SEQ ID NOs:4, 5, and 6; and FR regions that are human sequences (e.g., encoded by human light chain FR-encoding sequences).
  • a subject antibody comprises a light chain variable region that comprises, in order from N- terminus to C-terminus: a human light chain FR1; a CDRl comprising the amino acid sequence set forth in SEQ ID NO:4; a human light chain FR2; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; a human light chain FR3; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and a human light chain FR4.
  • a subject antibody comprises JAR 41 heavy chain CDRs and JAR 41 light chain CDRs in a single polypeptide chain, e.g., in some embodiments, a subject antibody is a scFv.
  • a subject antibody comprises, in order from N- terminus to C-terminus: a first amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDRl comprising the amino acid sequence set forth in SEQ ID NO: l; a second amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; a third amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; a fourth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDRl comprising the amino acid sequence set forth in SEQ ID:4; a fifth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; a sixth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR3 comprising the amino acid
  • a subject antibody can comprise a heavy chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence depicted in Figure 11A and set forth in SEQ ID NO: 18.
  • a subject antibody can comprise a heavy chain variable region comprising one, two, or three of the heavy chain complementarity determining regions (CDRs) having an amino acid sequence selected from one or more of SEQ ID NOs: l l, 12, and 13.
  • a subject antibody comprises a heavy chain variable region comprising one, two, or three of the heavy chain CDRs having an amino acid sequence selected from one or more of SEQ ID NOs: 11, 12, and 13; and FR regions that are human sequences (e.g., encoded by human heavy chain FR-encoding sequences).
  • a heavy chain variable region comprising one, two, or three of the heavy chain CDRs having an amino acid sequence selected from one or more of SEQ ID NOs: 11, 12, and 13; and FR regions that are human sequences (e.g., encoded by human heavy chain FR-encoding sequences).
  • a subject antibody comprises a heavy chain variable region that comprises, in order from N-terminus to C-terminus: a human heavy chain FR1; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11 ; a human heavy chain FR2; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12; a human heavy chain FR3; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; and a human heavy chain FR4.
  • a subject antibody can comprise a light chain variable region comprising an amino acid sequence that is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence depicted in Figure 11B and set forth in SEQ ID NO:20.
  • a subject antibody can comprise a light chain variable region comprising one, two, or three of the light chain CDRs having the amino acid sequence set forth in SEQ ID NOs: 14, 15, and 16.
  • a subject antibody comprises a light chain variable region comprising one, two, or three of the light chain CDRs having a polypeptide sequence selected from one or more of SEQ ID NOs: 14, 15, and 16; and FR regions that are human sequences (e.g., encoded by human light chain FR-encoding sequences).
  • a light chain variable region comprising one, two, or three of the light chain CDRs having a polypeptide sequence selected from one or more of SEQ ID NOs: 14, 15, and 16; and FR regions that are human sequences (e.g., encoded by human light chain FR-encoding sequences).
  • a subject antibody comprises a light chain variable region that comprises, in order from N-terminus to C-terminus: a human light chain FR1; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14; a human light chain FR2; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; a human light chain FR3; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 16; and a human light chain FR4.
  • a subject antibody comprises JAR 64 heavy chain CDRs and JAR 64 light chain CDRs in a single polypeptide chain, e.g., in some embodiments, a subject antibody is a scFv.
  • a subject antibody comprises, in order from N- terminus to C-terminus: a first amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11; a second amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12; a third amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13; a fourth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 14; a fifth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 15; a sixth amino acid sequence of from about 5 amino acids to about 25 amino acids in length; a CDR3 comprising the amino acid
  • the subject antibody does not contain one or more of the
  • CDRs of JAR 4 e.g., a subject anti-fHbp antibody will not include one or more of a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 of JAR 4, where JAR4 VH and VL CDR amino acid sequences are presented in Figure 12.
  • a subject anti-fHbp antibody does not include one, two, three, four, five, or all six of a VH CDR1, a VH CDR2, a VH CDR3, a VL CDR1, a VL CDR2, and a VL CDR3 of JAR 4, where JAR4 VH and VL CDR amino acid sequences are presented in Figure 12.
  • a subject antibody can include one or more linker regions, which can be a peptide or non-peptide linker.
  • the linker region can be from about 5 amino acids to about 50 amino acids in length, e.g., from about 5 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 35 aa, from about 35 aa to about 40 aa, from about 40 aa to about 45 aa, or from about 45 aa to about 50 aa in length.
  • Linkers suitable for use a subject antibody include “flexible linkers". If present, the linker molecules are generally of sufficient length to permit some flexible movement between linked regions. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules which can bind to polypeptides may be used in light of this disclosure.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Exemplary flexible linkers include glycine polymers (G) n , glycine- serine polymers (including, for example, (GS) n , GSGGS n (SEQ ID NO:47) and GGGS n (SEQ ID NO:48), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • Glycine and glycine- serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary flexible linkers include, but are not limited GGSG (SEQ ID NO:49), GGSGG (SEQ ID NO:50), GSGSG (SEQ ID NO:51), GSGGG (SEQ ID NO:52), GGGSG (SEQ ID NO:53), GSSSG (SEQ ID NO:54), and the like.
  • the ordinarily skilled artisan will recognize that design of a peptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
  • a subject antibody is "humanized.”
  • humanized antibody refers to an antibody comprising at least one chain comprising variable region framework residues substantially from a human antibody chain (referred to as the acceptor immunoglobulin or antibody) and at least one CDR substantially from a mouse antibody, (referred to as the donor immunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029 10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,585,089, U.S. Pat. No. 5,693,761, WO 90/07861, and U.S. Pat. No. 5,225,539.
  • the constant region(s), if present, can also be substantially or entirely from a human immunoglobulin.
  • a subject antibody comprises one or more JAR 41 or one or more JAR 64 CDRs and one or more FR regions from a human antibody.
  • Methods of making humanized antibodies are known in the art. See, e.g., U.S. Patent No. 7,256,273.
  • the substitution of mouse CDRs into a human variable domain framework can result in retention of their correct spatial orientation where, e.g., the human variable domain framework adopts the same or similar conformation to the mouse variable framework from which the CDRs originated.
  • This can be achieved by obtaining the human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable framework domains from which the CDRs were derived.
  • the heavy and light chain variable framework regions can be derived from the same or different human antibody sequences.
  • the human antibody sequences can be the sequences of naturally occurring human antibodies or can be consensus sequences of several human antibodies. See
  • the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody.
  • substitution of human amino acid residues with murine should be minimized, because introduction of murine residues increases the risk of the antibody eliciting a human-anti-mouse-antibody (HAMA) response in humans.
  • HAMA human-anti-mouse-antibody
  • Art-recognized methods of determining immune response can be performed to monitor a HAMA response in a particular patient or during clinical trials. Patients administered humanized antibodies can be given an immunogenicity assessment at the beginning and throughout the administration of said therapy.
  • the HAMA response is measured, for example, by detecting antibodies to the humanized therapeutic reagent, in serum samples from the patient using a method known to one in the art, including surface plasmon resonance technology (BIACORE) and/or solid-phase ELISA analysis.
  • BIACORE surface plasmon resonance technology
  • a subject humanized antibody does not substantially elicit a HAMA response in a human subject.
  • Certain amino acids from the human variable region framework residues are selected for substitution based on their possible influence on CDR conformation and/or binding to antigen.
  • the unnatural juxtaposition of murine CDR regions with human variable framework region can result in unnatural conformational restraints, which, unless corrected by substitution of certain amino acid residues, lead to loss of binding affinity.
  • the selection of amino acid residues for substitution can be determined, in part, by computer modeling.
  • Computer hardware and software for producing three-dimensional images of immunoglobulin molecules are known in the art.
  • molecular models are produced starting from solved structures for immunoglobulin chains or domains thereof.
  • the chains to be modeled are compared for amino acid sequence similarity with chains or domains of solved three-dimensional structures, and the chains or domains showing the greatest sequence similarity is/are selected as starting points for construction of the molecular model.
  • Chains or domains sharing at least 50% sequence identity are selected for modeling, and preferably those sharing at least 60%, 70%, 80%, or 90% sequence identity, or more than 90% identity (e.g., 95% identity, 98% identity, or 99% identity) are selected for modeling.
  • the solved starting structures are modified to allow for differences between the actual amino acids in the immunoglobulin chains or domains being modeled, and those in the starting structure.
  • the modified structures are then assembled into a composite immunoglobulin.
  • the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from one another and that bond lengths and angles are within chemically acceptable limits.
  • CDR and framework regions are as defined by Kabat, Sequences of Proteins of
  • Residues which are "adjacent to a CDR region" include amino acid residues in positions immediately adjacent to one or more of the CDRs in the primary sequence of the humanized immunoglobulin chain, for example, in positions immediately adjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia (See e.g., Chothia and Lesk JMB 196:901 (1987)), or a CDR as defined by Martin, supra.
  • amino acids are particularly likely to interact with the amino acids in the CDRs and, if chosen from the acceptor, to distort the donor CDRs and reduce affinity.
  • the adjacent amino acids may interact directly with the antigen (Amit et al., Science, 233:747 (1986)) and selecting these amino acids from the donor may be desirable to keep all the antigen contacts that provide affinity in the original antibody.
  • a subject antibody comprises scFv multimers.
  • a subject antibody is an scFv dimer (e.g., comprises two tandem scFv (scFv 2 )), an scFv trimer (e.g., comprises three tandem scFv (scFv 3 )), an scFv tetramer (e.g., comprises four tandem scFv (scFv 4 )), or is a multimer of more than four scFv (e.g., in tandem).
  • the scFv monomers can be linked in tandem via linkers of from about 2 amino acids to about 10 amino acids in length, e.g., 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa in length.
  • Suitable linkers include, e.g., (Gly) x , where x is an integer from 2 to 10. Other suitable linkers are those discussed above.
  • each of the scFv monomers in a subject scFV multimer is humanized, as described above.
  • a subject antibody comprises a constant region of an immunoglobulin (e.g., an Fc region).
  • the Fc region if present, can be a human Fc region. If constant regions are present, the antibody can contain both light chain and heavy chain constant regions. Suitable heavy chain constant region include CHI, hinge, CH2, CH3, and CH4 regions.
  • the antibodies described herein include antibodies having all types of constant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4.
  • An example of a suitable heavy chain Fc region is a human isotype IgGl Fc.
  • Light chain constant regions can be lambda or kappa.
  • a subject antibody e.g., a subject humanized antibody
  • Antibodies can be expressed as tetramers containing two light and two heavy chains, as separate heavy chains, light chains, as Fab, Fab' F(ab')2, and Fv, or as single chain antibodies in which heavy and light chain variable domains are linked through a spacer.
  • a subject antibody comprises a free thiol (-SH) group at the carboxyl terminus, where the free thiol group can be used to attach the antibody to a second polypeptide (e.g., another antibody, including a subject antibody), a scaffold, a carrier, etc.
  • a second polypeptide e.g., another antibody, including a subject antibody
  • a subject antibody comprises one or more non-naturally occurring amino acids.
  • the non-naturally encoded amino acid comprises a carbonyl group, an acetyl group, an aminooxy group, a hydrazine group, a hydrazide group, a semicarbazide group, an azide group, or an alkyne group. See, e.g., U.S. Patent No. 7,632,924 for examples of suitable non-naturally occurring amino acids.
  • Inclusion of a non-naturally occurring amino acid can provide for linkage to a polymer, a second polypeptide, a scaffold, etc.
  • a subject antibody linked to a water-soluble polymer can be made by reacting a water-soluble polymer (e.g., PEG) that comprises a carbonyl group with a subject fHbp-binding subject antibody that comprises a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • a subject antibody linked to a water-soluble polymer can be made by reacting a subject antibody that comprises an alkyne- containing amino acid with a water-soluble polymer (e.g., PEG) that comprises an azide moiety; in some embodiments, the azide or alkyne group is linked to the PEG molecule through an amide linkage.
  • a "non-naturally encoded amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used
  • non-naturally encoded amino acid synonymously with the term “non-naturally encoded amino acid” are “non-natural amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof.
  • non-naturally encoded amino acid also includes, but is not limited to, amino acids that occur by modification (e.g. post-translational
  • a naturally encoded amino acid including but not limited to, the 20 common amino acids or pyrolysine and selenocysteine
  • non-naturally- occurring amino acids include, but are not limited to, N-acetylglucosaminyl-L-serine, N- acetylglucosaminyl-L-threonine, and O -phosphotyrosine.
  • a subject antibody is linked (e.g., covalently linked) to a polymer (e.g., a polymer other than a polypeptide).
  • a polymer e.g., a polymer other than a polypeptide.
  • Suitable polymers include, e.g.,
  • Suitable polymers include synthetic polymers and naturally-occurring polymers. Suitable polymers include, e.g., substituted or unsubstituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymers or branched or unbranched polysaccharides, e.g. a homo- or hetero-polysaccharide. Suitable polymers include, e.g., ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or by the trade name EVAL); polybutylmethacrylate;
  • PEO/PLA poly(ethylene oxide)-poly(lactic acid)
  • polyurethanes silicones; polyesters; polyolefins; polyisobutylene and ethylene- alphaolef in copolymers; acrylic polymers and copolymers; vinyl halide polymers and copolymers, such as polyvinyl chloride; polyvinyl ethers, such as polyvinyl methyl ether; polyvinylidene halides, such as polyvinylidene fluoride and polyvinylidene chloride; polyacrylonitrile; polyvinyl ketones; polyvinyl aromatics, such as polystyrene; polyvinyl esters, such as polyvinyl acetate; copolymers of vinyl monomers with each other and olefins, such as ethylene-methyl
  • methacrylate copolymers acrylonitrile-styrene copolymers, ABS resins, and ethylene-vinyl acetate copolymers
  • polyamides such as Nylon 66 and polycaprolactam
  • alkyd resins alkyd resins
  • polycarbonates polyoxymethylenes; polyimides; polyethers; epoxy resins; polyurethanes; rayon; rayon-triacetate; cellulose; cellulose acetate; cellulose butyrate; cellulose acetate butyrate;
  • poly(ethylene glycol); and carboxymethyl cellulose are examples of poly(ethylene glycol); and carboxymethyl cellulose.
  • Suitable synthetic polymers include unsubstituted and substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol), and derivatives thereof, e.g., substituted poly(ethyleneglycol) such as methoxypoly(ethyleneglycol), and derivatives thereof.
  • Suitable naturally-occurring polymers include, e.g., albumin, amylose, dextran, glycogen, and derivatives thereof.
  • Suitable polymers can have an average molecular weight in a range of from 500
  • the PEG or methoxypoly(ethyleneglycol) polymer can have a molecular weight in a range of from about 0.5 kiloDaltons (kDa) to 1 kDa, from about 1 kDa to 5 kDa, from 5 kDa to 10 kDa, from 10 kDa to 25 kDa, from 25 kDa to 40 kDa, or from 40 kDa to 60 kDa.
  • kDa kiloDaltons
  • a subject antibody is covalently linked to a PEG polymer.
  • a subject scFv multimer can be covalently linked to a PEG polymer. See, e.g., Albrecht et al. (2006) J. Immunol. Methods 310: 100. Methods and reagents suitable for PEGylation of a protein are well known in the art and may be found in, e.g., U.S. Pat. No. 5,849,860.
  • PEG suitable for conjugation to a protein is generally soluble in water at room temperature, and has the general formula R(0-CH 2 -CH 2 ) n O-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.
  • R is hydrogen or a protective group such as an alkyl or an alkanol group
  • n is an integer from 1 to 1000.
  • R is a protective group, it generally has from 1 to 8 carbons.
  • the PEG conjugated to the subject antibody can be linear.
  • the PEG conjugated to the subject protein may also be branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, "star-PEG's" and multi-armed PEG's such as those described in
  • a subject antibody can be glycosylated, e.g., comprise a covalently linked carbohydrate or polysaccharide moiety.
  • Glycosylation of antibodies is typically either N-linked or O-linked.
  • N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.
  • O-linked glycosylation refers to the attachment of a sugar such as N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, e.g., attachment to a serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used.
  • a sugar such as N-acetylgalactosamine, galactose, or xylose
  • a hydroxyamino acid e.g., attachment to a serine or threonine, although 5- hydroxyproline or 5-hydroxylysine may also be used.
  • Addition of glycosylation sites to an antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites).
  • the alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).
  • removal of glycosylation sites can be accomplished by amino acid alteration within the native glycosylation sites of an antibody.
  • a subject antibody will in some embodiments comprise a "radiopaque" label, e.g. a label that can be easily visualized using for example x-rays.
  • Radiopaque materials are well known to those of skill in the art. The most common radiopaque materials include iodide, bromide or barium salts. Other radiopaque materials are also known and include, but are not limited to organic bismuth derivatives (see, e.g., U.S. Pat. No. 5,939,045), radiopaque multiurethanes (see U.S. Pat. No. 5,346,981), organobismuth composites (see, e.g., U.S. Pat. No. 5,256,334), radiopaque barium multimer complexes (see, e.g., U.S. Pat. No. 4,866,132), and the like.
  • a subject antibody can be covalently linked to a second moiety (e.g., a lipid, a polypeptide other than a subject antibody, a synthetic polymer, a carbohydrate, and the like) using for example, glutaraldehyde, a homobifunctional cross-linker, or a heterobifunctional cross-linker.
  • Glutaraldehyde cross-links polypeptides via their amino moieties.
  • Homobifunctional cross-linkers e.g., a homobifunctional imidoester, a homobifunctional N- hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl reactive cross-linker
  • a homobifunctional imidoester e.g., a homobifunctional N- hydroxysuccinimidyl (NHS) ester, or a homobifunctional sulfhydryl reactive cross-linker
  • Homobifunctional NHS ester and imido esters cross-link amine containing polypeptides. In a mild alkaline pH, imido esters react only with primary amines to form imidoamides, and overall charge of the cross-linked polypeptides is not affected.
  • Homobifunctional sulfhydryl reactive cross-linkers includes bismaleimidhexane (BMH), l,5-difluoro-2,4-dinitrobenzene (DFDNB), and l,4-di-(3',2'-pyridyldithio) propinoamido butane (DPDPB).
  • BMH bismaleimidhexane
  • DFDNB l,5-difluoro-2,4-dinitrobenzene
  • DPDPB l,4-di-(3',2'-pyridyldithio) propinoamido butane
  • Heterobifunctional cross-linkers have two or more different reactive moieties
  • heterobifunctional haloacetyl cross-linkers are available, as are pyridyl disulfide cross -linkers.
  • Carbodiimides are a classic example of heterobifunctional cross-linking reagents for coupling carboxyls to amines, which results in an amide bond.
  • a subject antibody can be immobilized on a solid support.
  • Suitable supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracytes, wells of reaction trays (e.g., multi-well plates), plastic tubes, etc.
  • a solid support can comprise any of a variety of substances, including, e.g., glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • Solid supports can be soluble or insoluble, e.g., in aqueous solution.
  • a suitable solid support is generally insoluble in an aqueous solution.
  • a subject antibody will in some embodiments comprise a detectable label.
  • Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable include, but are not limited to, magnetic beads (e.g. DynabeadsTM), fluorescent dyes (e.g., fluorescein
  • radiolabels e.g., 3 H, 125 I, 35 S, 14 C, or 32 P
  • enzymes e.g., horse radish peroxidase, alkaline phosphatase, luciferase, and others commonly used in an enzyme-linked immunosorbent assay (ELISA)
  • colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • a subject antibody comprises a contrast agent or a radioisotope, where the contrast agent or radioisotope is one that is suitable for use in imaging, e.g., imaging procedures carried out on humans.
  • labels include radioisotope such as 125 I (iodine), 18 F (fluorine), 99 Tc (technetium), lu In (indium), and 67 Ga (gallium), and contrast agent such as gadolinium (Gd), dysprosium, and iron.
  • Radioactive Gd isotopes ( 153 Gd) also are available and suitable for imaging procedures in non-human mammals.
  • a subject antibody can be labeled using standard techniques.
  • a subject antibody can be iodinated using chloramine T or l,3,4,6-tetrachloro-3a,6a-dephenylglycouril.
  • fluorination fluorine is added to a subject antibody during the synthesis by a fluoride ion displacement reaction. See, MuUer-Gartner, H., TIB Tech., 16: 122-130 (1998) and Saji, H., Crit. Rev. Ther. Drug Carrier Syst, 16(2):209-244 (1999) for a review of synthesis of proteins with such radioisotopes.
  • a subject antibody can also be labeled with a contrast agent through standard techniques.
  • a subject antibody can be labeled with Gd by conjugating low molecular Gd chelates such as Gd diethylene triamine pentaacetic acid (GdDTPA) or Gd tetraazacyclododecanetetraacetic (GdDOTA) to the antibody.
  • GdDTPA Gd diethylene triamine pentaacetic acid
  • GdDOTA Gd tetraazacyclododecanetetraacetic
  • a subject antibody can be labeled with Gd by, for example, conjugating polylysine-Gd chelates to the antibody. See, for example, Curtet et al., Invest. Radiol., 33(10):752-761 (1998).
  • a subject antibody can be labeled with Gd by incubating paramagnetic polymerized liposomes that include Gd chelator lipid with avidin and biotinylated antibody. See, for example, Sipkins et al., Nature Med., 4:623-626 (1998).
  • Suitable fluorescent proteins that can be linked to a subject antibody include, but are not limited to, a green fluorescent protein from Aequoria victoria or a mutant or derivative thereof e.g., as described in U.S. Patent No. 6,066,476; 6,020,192; 5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445; 5,874,304; e.g., Enhanced GFP, many such GFP which are available commercially, e.g., from Clontech, Inc.; a red fluorescent protein; a yellow fluorescent protein; any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973; and the like.
  • a subject antibody will in some embodiments be linked to (e.g., covalently or non-covalently linked) a fusion partner, e.g., a ligand; an epitope tag; a peptide; a protein other than an antibody; and the like.
  • a fusion partner e.g., a ligand; an epitope tag; a peptide; a protein other than an antibody; and the like.
  • Suitable fusion partners include peptides and polypeptides that confer enhanced stability in vivo (e.g., enhanced serum half-life); provide ease of purification, e.g., (His) n , e.g., 6His, and the like; provide for secretion of the fusion protein from a cell;
  • GST glutathione-S-transferase
  • HA hemagglutinin
  • CYPYDVPDYA SEQ ID NO:55
  • FLAG e.g., DYKDDDDK
  • SEQ ID NO:56 e.g., DYKDDDDK
  • c-myc e.g., CEQKLISEEDL; SEQ ID NO:57
  • a detectable signal e.g., an enzyme that generates a detectable product (e.g., ⁇ -galactosidase, luciferase), or a protein that is itself detectable, e.g., a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, etc.
  • multimerization e.g., a multimerization domain such as an Fc portion of an immunoglobulin; and the like.
  • the fusion may also include an affinity domain, including peptide sequences that can interact with a binding partner, e.g., such as one immobilized on a solid support, useful for identification or purification.
  • a binding partner e.g., such as one immobilized on a solid support
  • Consecutive single amino acids, such as histidine when fused to a protein, can be used for one-step purification of the fusion protein by high affinity binding to a resin column, such as nickel sepharose.
  • affinity domains include His5 (HHHHH) (SEQ ID NO:58), HisX6 (HHHHHH) (SEQ ID NO:59), c-myc (EQKLISEEDL) (SEQ ID NO:60), Flag (DYKDDDDK) (SEQ ID NO:61), StrepTag (WSHPQFEK) (SEQ ID NO:62), hemagglutinin, e.g., HA Tag (YPYDVPDYA; SEQ ID NO:63), glutathinone-S-transferase (GST), thioredoxin, cellulose binding domain, RYIRS (SEQ ID NO:64), Phe-His-His-Thr (SEQ ID NO:65), chitin binding domain, S-peptide, T7 peptide, SH2 domain, C-end RNA tag, WEAAAREACCRECCARA (SEQ ID NO:66), metal binding domains, e.g., zinc binding domains or calcium binding domains such as those from calcium-binding proteins
  • a subject antibody is modified to include a carbohydrate moiety, where the carbohydrate moiety can be covalently linked to the antibody.
  • a subject antibody is modified to include a lipid moiety, where the lipid moiety can be covalently linked to the antibody.
  • Suitable lipid moieties include, e.g., an N-fatty acyl group such as N-lauroyl, N-oleoyl, etc.; a fatty amine such as dodecyl amine, oleoyl amine, etc.; a C3-C16 long-chain aliphatic lipid; and the like. See, e.g., U.S. Pat. No. 6,638,513).
  • a subject antibody is incorporated into a liposome.
  • An anti-fHbp antibody of the present disclosure can be produced by any known method, e.g., conventional synthetic methods for protein synthesis; recombinant DNA methods; etc.
  • a subject antibody is a single chain polypeptide
  • it can be synthesized using standard chemical peptide synthesis techniques.
  • the synthesis may proceed via liquid-phase or solid-phase.
  • Solid phase polypeptide synthesis SPPS
  • Fmoc and Boc Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing a subject antibody.
  • Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide
  • nucleic acids encoding light and heavy chain variable regions are inserted into expression vectors.
  • the light and heavy chains can be cloned in the same or different expression vectors.
  • the DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.
  • Expression control sequences include, but are not limited to, promoters (e.g., naturally- associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.
  • the expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells (e.g., COS or CHO cells). Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the antibodies.
  • nucleic acid sequences can encode each immunoglobulin amino acid sequence.
  • the desired nucleic acid sequences can be produced by de novo solid-phase DNA synthesis or by polymerase chain reaction (PCR) mutagenesis of an earlier prepared variant of the desired polynucleotide.
  • Oligonucleotide - mediated mutagenesis is an example of a suitable method for preparing substitution, deletion and insertion variants of target polypeptide DNA. See Adelman et al., DNA 2: 183 (1983). Briefly, the target polypeptide DNA is altered by hybridizing an oligonucleotide encoding the desired mutation to a single- stranded DNA template. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that incorporates the oligonucleotide primer, and encodes the selected alteration in the target polypeptide DNA.
  • Suitable expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers (e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance) to permit detection of those cells transformed with the desired DNA sequences.
  • selection markers e.g., ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance or neomycin resistance
  • Escherichia coli is an example of a prokaryotic host cell that can be used for cloning a subject antibody-encoding polynucleotide.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • yeast Other microbes, such as yeast, are also useful for expression. Saccharomyces
  • yeast host cells e.g., S. cerevisiae
  • suitable vectors having expression control sequences (e.g., promoters), an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • mammalian cells e.g., mammalian cells grown in in vitro cell culture
  • the polypeptides of the present invention e.g., polynucleotides encoding immunoglobulins or fragments thereof.
  • Suitable mammalian host cells include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, and transformed B-cells or hybridomas.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Suitable expression control sequences are promoters derived from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Co et al., J. Immunol. 148: 1149 (1992).
  • the whole antibodies, their dimers, individual light and heavy chains, or other forms of a subject antibody can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, high performance liquid
  • a subject antibody can be substantially pure, e.g., at least about 80% to 85% pure, at least about 85% to 90% pure, at least about 90% to 95% pure, or 98% to 99%, or more, pure, e.g., free from contaminants such as cell debris, macromolecules other than a subject antibody, etc.
  • a subject antibody composition can comprise, in addition to a subject antibody, one or more of: a salt, e.g., NaCl, MgCl 2 , KC1, MgS0 4 , etc.; a buffering agent, e.g., a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N- Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as T
  • nucleic acids comprising nucleotide sequences encoding a subject anti-fHbp antibody.
  • a nucleotide sequence encoding a subject antibody can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g., a cell that is genetically modified to synthesize the encoded antibody).
  • the nucleic acid is present within an expression vector.
  • Suitable promoter and enhancer elements are known in the art.
  • suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc.
  • suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements;
  • cytomegalovirus immediate early promoter herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters.
  • a suitable promoter is a constitutive promoter such as an ADHl promoter, a PGKl promoter, an ENO promoter, a PYKl promoter and the like; or a regulatable promoter such as a GAL1 promoter, a GAL10 promoter, an ADH2 promoter, a PH05 promoter, a CUP1 promoter, a GAL7 promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS 3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia).
  • a constitutive promoter such as an ADHl promoter, a PGKl promoter, an ENO promoter
  • Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter ⁇ see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. BacterioL, 1991: 173(1): 86-93;
  • nirB promoter e.g., Dunstan et al. (1999) Infect. Immun. 67:5133- 5141; McKelvie et al. (2004) Vaccine 22:3243-3255; and Chatfield et al. (1992) Biotechnol. 10:888-892
  • a sigma70 promoter e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos.
  • a stationary phase promoter e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPI-2 ⁇ see, e.g., W096/17951); an actA promoter ⁇ see, e.g., Shetron-Rama et al. (2002) Infect. Immun. 70: 1087-1096); an rpsM promoter ⁇ see, e.g., Valdivia and Falkow (1996). Mol. Microbiol.
  • Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and PLambda.
  • Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, for example, deBoer et al. (1983) Proc. Natl. Acad. Sci. U.S.A. 80:21-25).
  • a lactose promoter operator Lacl repressor protein changes conformation when contacted with lactose, thereby preventing the Lacl repressor protein from binding to the operator
  • TrpR repressor protein when complexed with tryptophan, TrpR
  • a nucleotide sequence encoding a subject antibody can be present in an expression vector and/or a cloning vector. Where a subject antibody comprises two separate polypeptides, nucleotide sequences encoding the two polypeptides can be cloned in the same or separate vectors.
  • An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector.
  • Bacterial pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden).
  • Eukaryotic pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).
  • Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins.
  • a selectable marker operative in the expression host may be present.
  • Suitable expression vectors include, but are not limited to, viral vectors (e.g.
  • viral vectors based on vaccinia virus; poliovirus; adenovirus see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther
  • a retroviral vector e.g., Murine Leukemia Virus, s
  • a subject nucleic acid comprises a nucleotide sequence encoding a subject antibody.
  • a subject nucleic acid can comprise a nucleotide sequence encoding heavy- and light-chain anti-fHbp CDRs.
  • a subject nucleic acid comprises a nucleotide sequence encoding heavy- and light-chain JAR 41 or JAR 64 CDRs, where the CDR- encoding sequences are interspersed with FR-encoding nucleotide sequences.
  • the FR-encoding nucleotide sequences are human FR-encoding nucleotide sequences.
  • the present disclosure provides isolated genetically modified host cells (e.g., in vitro cells) that are genetically modified with a subject nucleic acid.
  • a subject isolated genetically modified host cell can produce a subject antibody.
  • Suitable host cells include eukaryotic host cells, such as a mammalian cell, an insect host cell, a yeast cell; and prokaryotic cells, such as a bacterial cell.
  • eukaryotic host cells such as a mammalian cell, an insect host cell, a yeast cell
  • prokaryotic cells such as a bacterial cell.
  • Introduction of a subject nucleic acid into the host cell can be effected, for example by calcium phosphate precipitation, DEAE dextran mediated transfection, liposome-mediated transfection,
  • Suitable mammalian cells include primary cells and immortalized cell lines.
  • Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like.
  • Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No.
  • HeLa cells e.g., American Type Culture Collection (ATCC) No. CCL-2
  • CHO cells e.g., ATCC Nos. CRL9618, CCL61, CRL9096
  • 293 cells e.g., ATCC No. CRL
  • CRL1721) COS cells
  • COS-7 cells ATCC No. CRL1651
  • RATI cells mouse L cells
  • mouse L cells ATCC No. CCLI.3
  • human embryonic kidney (HEK) cells ATCC No. CRL1573
  • HLHepG2 cells and the like.
  • Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fus
  • Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., and the like. See, e.g., Carrier et al. (1992) J. Immunol. 148: 1176-1181; U.S. Patent No. 6,447,784; and Sizemore et al. (1995) Science 270:299-302.
  • Salmonella strains which can be employed include, but are not limited to, Salmonella typhi and S. typhimurium.
  • Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae.
  • the laboratory strain is one that is non-pathogenic.
  • suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita,
  • the host cell is Escherichia coli.
  • an anti-fHbp antibody of the present disclosure can be used in various detection methods.
  • an anti-fHbp antibody of the present disclosure can be used to detect the presence and/or level of fHbp in a sample (e.g., free fHbp (e.g., soluble fHbp); or fHbp on the surface of a bacterial cell, e.g., a Neisseria meningitidis cell).
  • a sample e.g., free fHbp (e.g., soluble fHbp); or fHbp on the surface of a bacterial cell, e.g., a Neisseria meningitidis cell.
  • an N. meningitidis cell that is being considered as a target for an fHbp vaccine can be tested for the level of fHbp on the surface of the cell.
  • a test sample is contacted with an anti-fHbp antibody of the present disclosure; and binding of the anti-fHbp antibody to an epitope in the sample is detected.
  • the level of fHbp present in the test sample can be determined by the level of bound antibody.
  • the level of fHbp can be compared to a level of a reference strain, e.g., a level of fHbp of N. meningitidis strain H44/76.
  • test cell has a level of fHbp within about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 50% of a level of fHbp detectable on the cell surface of a reference strain, where the reference strain expresses a level of fHbp that renders it susceptible to bactericidal activity of an anti-fHbp antibody, then the test cell is identified as one that is a target for an fHbp vaccine.
  • the test sample can be a clinical isolate (where the isolate includes a whole N. meningitidis cell); a solubilized N. meningitidis cell; a fraction of a N. meningitidis cell; an outer membrane vesicle (OMV); etc.
  • a test sample includes a sample that has been manipulated in any way after its procurement, such as by treatment with reagents, solubilization, or enrichment for certain components, such as proteins.
  • Suitable assay formats include, but are not limited to, quantitative protein blot ("Western” blot); capture enzyme linked immunosorbent assay (ELISA); a flow cytometric assay; and the like.
  • the antibody used in a subject detection method is immobilized on an insoluble (e.g., a solid) support.
  • Suitable supports are well known in the art and comprise, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloid metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, nylon membranes, sheets, duracytes, wells of reaction trays (e.g., multi-well plates), test strips, plastic tubes, etc.
  • a solid support can comprise any of a variety of substances, including, e.g., glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene,
  • Solid supports can be soluble or insoluble, e.g., in aqueous solution. In some embodiments, a suitable solid support is generally insoluble in an aqueous solution.
  • the anti-fHbp antibody used in a detection method of the present disclosure can comprise a detectable label.
  • an anti-fHbp antibody does not include a detectable label, but is instead detected using a second antibody that binds the anti-fHbp antibody, where the second antibody comprises a detectable label.
  • Suitable detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Suitable include, but are not limited to, magnetic beads (e.g.
  • DynabeadsTM fluorescent dyes (e.g., fluorescein isothiocyanate, texas red, rhodamine, a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, and the like), radiolabels (e.g., 3 H, 125 I, 35 S, 14 C, or 32 P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase, luciferase, and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.
  • fluorescent dyes e.g., fluorescein isothiocyanate, texas red, rhodamine, a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, and the like
  • radiolabels e.g., 3 H, 125 I, 35 S, 14 C, or
  • a subject detection method involves contacting a test N.
  • meningitidis strain e.g., a test sample comprising a N. meningitidis strain
  • an anti-fHbp antibody of the present disclosure e.g., a test sample comprising a N. meningitidis strain
  • detecting binding of the antibody to an fHbp epitope present on the surface of the cell e.g., binding of a subject anti-fHbp antibody to live bacteria can be assessed by flow cytometry.
  • Bacterial cells are incubated with a subject anti- fHbp antibody, which antibody can be detectably labeled, or which antibody can be detected with a second labeled antibody (e.g., a labeled anti-IgG antibody, as shown in Figure 7).
  • the bacteria are washed; and bound anti-fHbp antibody is detected.
  • a detection method of the present disclosure can detect the level of fHbp present in a test sample.
  • the detection method can be used, e.g., where the fHbp is not attached to a cell, e.g., where the fHbp is soluble.
  • the detection method can also be used with whole cells, e.g., N. meningitidis expressing fHbp on the cell surface.
  • assays to detect fHbp can involve use of a subject anti-fHbp antibody immobilized on a support (e.g., anti-fHbp immobilized on the well of a microtiter plate, on a test strip, etc.; see for example, Donnelly et al. (2010) Proc. Natl. Acad. Sci. USA
  • a test sample that contains fHbp (e.g., free fHbp, or bacteria expressing fHbp) is contacted with the immobilized anti-fHbp antibody and incubated for an amount of time sufficient to allow for antibody binding; immobilized anti-fHbp antibody that binds fHbp forms an immobilized anti-fHbp antibody/fHbp complex. After washing the immobilized anti-fHbp antibody, bound fHbp is detected with a second specific anti-fHbp antibody that recognizes an epitope on fHbp that is different that the immobilized antibody.
  • fHbp e.g., free fHbp, or bacteria expressing fHbp
  • the second antibody may contain a labeled component, or may be detected with a third labeled antibody or antiserum that binds specifically to the second antibody.
  • a polyclonal antiserum to fHbp may be used (e.g., serum from a rabbit immunized with fHbp) to detect bound fHbp, which can be detected with a labeled antibody (e.g., goat anti-rabbit anti-serum).
  • a labeled antibody e.g., goat anti-rabbit anti-serum.
  • the amount of fHbp present in the immobilized anti-fHbp antibody/fHbp complex is determined by the amount of label detected.
  • the detection of fHbp on whole cells can be carried out using a monoclonal first antibody (such as JAR 41) as a "capture antibody"; the detection antibody may be a monoclonal antibody that recognizes a different epitope from the epitope recognized by the first antibody, or may be a polyclonal anti-serum (such as that from a fHbp-immunized rabbit).
  • a monoclonal first antibody such as JAR 41
  • the detection antibody may be a monoclonal antibody that recognizes a different epitope from the epitope recognized by the first antibody, or may be a polyclonal anti-serum (such as that from a fHbp-immunized rabbit).
  • two different antibodies are used.
  • the assay detects fHbp on a whole cell
  • the assay involves use of a first antibody comprising CDRs derived from JAR 41, and a second antibody comprising CDRs derived from JAR 64.
  • the first antibody can comprise CDRs derived from JAR 64; and the second antibody can comprise CDRs derived from JAR 41.
  • the first antibody (the "capture antibody") is immobilized on a solid support.
  • a test sample that includes fHbp is contacted with the first antibody, such that the fHbp in the test sample binds to the first antibody, forming an immobilized first antibody/fHbp complex.
  • the second antibody (the "detection antibody"), which can include a detectable label, is then contacted with the immobilized first antibody/fHbp complex. Any fHbp present in the test sample is detected by the second antibody.
  • the second antibody can be labeled directly or indirectly.
  • Direct labels include moieties that are bound directly to the second antibody, such that the second antibody itself comprises a detectable label.
  • Indirect labels include secondary antibodies that comprise a detectable label, where secondary antibodies include, e.g., an antibody that will bind the second antibody. In this assay method, one or more optional washing steps can be included.
  • a subject detection method involves detection of fHbp, where the fHbp (either as a whole N. meningitidis cell or not associated with a cell) is immobilized on an insoluble support.
  • an N. meningitidis cell can be immobilized on an insoluble support; or a soluble fHbp can be immobilized on an insoluble support.
  • the immobilized fHbp is contacted with a subject anti-fHbp antibody, such that the anti-fHbp antibody binds the immobilized fHbp, forming an immobilized fHbp/anti-fHbp complex.
  • the anti-fHbp antibody can be labeled directly or indirectly.
  • Direct labels include moieties that are bound directly to the antibody, such that the antibody itself comprises a detectable label.
  • Indirect labels include secondary antibodies that comprise a detectable label, where secondary antibodies include, e.g., an antibody that will bind the anti-fHbp antibody. In this assay method, one or more optional washing steps can be included.
  • a detection method of the present disclosure can include one or more controls, e.g., a negative control that includes an irrelevant antibody (e.g. an antibody that does not bind fHbp).
  • a negative control that includes an irrelevant antibody (e.g. an antibody that does not bind fHbp).
  • a subject detection method allows one to predict the susceptibility of an N.
  • meningitidis strain to an fHbp vaccine.
  • a strain that expresses high levels of fHbp on the cell surface is expected to be more susceptible to anti-fHbp bactericidal activity than a strain with low levels of fHbp expression.
  • a subject detection method thus allows one to predict whether a given N. meningitidis strain will be susceptible to antibodies elicited by different fHbp vaccines.
  • An anti-fHbp antibody of the present disclosure can be used to treat a Neisseria meningitidis infection.
  • An anti-fHbp antibody of the present disclosure in combination with at least one additional anti-fHbp antibody, exhibits bactericidal activity toward N. meningitidis.
  • a treatment method of the present disclosure generally involves administering an effective amount of a subject antibody, alone (e.g., in monotherapy) or in combination (e.g., in
  • combination therapy with one or more additional therapeutic agents and/or one or more additional anti-fHbp antibodies.
  • an effective amount of a subject antibody is an amount that, alone or in combination with at least a second antibody, reduces bacteremia (e.g., reduces the number of N. meningitidis bacteria in the blood) in an individual by at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%.
  • bacteremia e.g., reduces the number of N. meningitidis bacteria in the blood
  • a subject therapeutic method may find use, e.g., in the treatment of individuals with terminal complement deficiencies who have increased risk for acquiring meningococcal infection and who do develop chronic meningococcal infection.
  • Combinations of anti-fHbp antibodies that are suitable for use in a subject treatment method include, e.g.: a) an antibody comprising JAR 41 CDRs; and b) a second anti- fHbp antibody that does not by itself exhibit bactericidal toward N. meningitidis.
  • Combinations of anti-fHbp antibodies that are suitable for use in a subject treatment method also include, e.g.: a) a antibody comprising JAR 64 CDRs; and b) a second anti-fHbp antibody that does not by itself exhibit bactericidal toward N. meningitidis.
  • the combination of the two antibodies provides for bactericidal activity toward N. meningitidis.
  • the second anti-fHbp antibody that does not by itself exhibit bactericidal toward N. meningitidis can be JAR5, JAR63, and the like.
  • the first antibody can be an antibody comprising JAR 41 CDRs (e.g., a JAR 41 antibody); and the second antibody can be a JAR 5 antibody.
  • JAR 5 JAR 63, mAb 502 have been tested and are positive for combined bactericidal activity with JAR 41 against strain H44/76.
  • JAR 11 and JAR 13 have combined bactericidal activity with JAR 41 against strain 8047, which is of a different variant group (variant group 2).
  • an anti-fHbp antibody can be administered to the host using any convenient means capable of resulting in the desired therapeutic effect.
  • the antibody can be incorporated into a variety of formulations for therapeutic administration.
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
  • Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985.
  • the composition or formulation to be administered will, in any event, contain a quantity of a subject antibody adequate to achieve the desired state in the subject being treated.
  • compositions such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • compositions comprising a subject antibody are prepared by mixing the antibody having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents.
  • Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine,
  • polypeptides such as polypeptides
  • proteins such as gelatin or serum albumin
  • chelating agents such as EDTA
  • sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-Methylglucosamine, galactosamine, and neuraminic acid
  • non-ionic surfactants such as Tween, Brij Pluronics, Triton-X or polyethylene glycol (PEG).
  • the pharmaceutical composition may be in a liquid form, a lyophilized form or a liquid form reconstituted from a lyophilized form, wherein the lyophilized preparation is to be reconstituted with a sterile solution prior to administration.
  • the standard procedure for reconstituting a lyophilized composition is to add back a volume of pure water (typically equivalent to the volume removed during lyophilization); however solutions comprising antibacterial agents may be used for the production of pharmaceutical compositions for parenteral administration; see also Chen (1992) Drug Dev Ind Pharm 18, 1311-54.
  • Exemplary antibody concentrations in a subject pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL.
  • An aqueous formulation of the antibody may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the antibody formulation to modulate the tonicity of the formulation.
  • exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • isotonic denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 nM.
  • a surfactant may also be added to the antibody formulation to reduce aggregation of the formulated antibody and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • Suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188TM.
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
  • Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v.
  • a lyoprotectant may also be added in order to protect the labile active ingredient
  • lyoprotectants include sugars (including glucose and sucrose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 nM.
  • a subject formulation includes a subject antibody, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at
  • concentrations ranging from about 0.001 to about 2% (w/v).
  • a subject formulation can be a liquid or lyophilized formulation suitable for parenteral administration, and can comprise: about 1 mg/mL to about 200 mg/mL of a subject antibody; about 0.001 % to about 1 % of at least one surfactant; about 1 mM to about 100 mM of a buffer; optionally about 10 mM to about 500 mM of a stabilizer; and about 5 mM to about 305 mM of a tonicity agent; and has a pH of about 4.0 to about 7.0.
  • a subject parenteral formulation is a liquid or lyophilized formulation comprising: about 1 mg/mL to about 200 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L-histidine; and 250 mM Sucrose; and has a pH of 5.5.
  • a subject parenteral formulation comprises a lyophilized formulation comprising: 1) 15 mg/mL of a subject antibody; 0.04% Tween 20 w/v; 20 mM L- histidine; and 250 mM sucrose; and has a pH of 5.5; or 2) 75 mg/mL of a subject antibody;
  • a subject parenteral formulation is a liquid formulation comprising: 1) 7.5 mg/mL of a subject antibody; 0.022% Tween 20 w/v; 120 mM L-histidine; and 250 125 mM sucrose; and has a pH of 5.5; or 2) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 3) 37.5 mg/mL of a subject antibody; 0.01% Tween 20 w/v; 10 mM L-histidine; and 125 mM sucrose; and has a pH of 5.5; or 4) 37.5 mg/mL of a subject antibody; 0.02% Tween 20 w/v; 10 mM L-histidine; 125 mM trehalose; and has a pH of 5.5; or 5) 37.5 mg/mL of a subject antibody;
  • a suitable dosage of an anti-fHbp antibody can be determined by an attending physician or other qualified medical personnel, based on various clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular antibody to be administered, sex of the patient, time, and route of administration, general health, and other drugs being administered concurrently.
  • a subject antibody (or combination of antibodies) may be administered in amounts between 1 ng/kg body weight and 20 mg/kg body weight per dose, e.g. between 0.1 mg/kg body weight to 10 mg/kg body weight, e.g.
  • the regimen is a continuous infusion, it can also be in the range of 1 ⁇ g to 10 mg per kilogram of body weight per minute.
  • dose levels can vary as a function of the specific antibody, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given antibody are readily determinable by those of skill in the art by a variety of means.
  • a subject antibody is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • a therapeutic method of the present disclosure can be carried out on an individual who has or is at risk of a N. meningitidis infection.
  • the individual has been treated for the N. meningitidis infection, but has not responded to the treatment.
  • an individual with an immunodeficiency such as terminal complement deficiency, may have a higher risk for developing recurrent or chronic N. meningitidis infection and may benefit from prophylactic therapy using a subject monoclonal antibody.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal (ly); s.c, subcutaneous(ly); and the like.
  • EXAMPLE 1 CHARACTERIZATION OF ANTI-FHBP MONOCLONAL ANTIBODIES
  • JAR 64 were generated.
  • JAR 41 was generated from a human factor H (fH) transgenic BALB/c mouse immunized with a recombinant fHbp ID 1 (variant group 1).
  • the recombinant fHbp ID 1 (v. l) binds to fH.
  • JAR 64 was generated from a human fH transgenic mouse immunized with a recombinant mutant of fHbp ID 1 (v.l).
  • arginine at position 41 (R41) is replaced by serine.
  • This mutant fHbp does not bind to fH as the R41S mutation eliminates binding to fH (Beernink et al. (2011) J Immunol. 186: 3606-14).
  • fH transgenic mice and mutant fHbp with the R41S mutation are described in U.S. Patent Publication No. 2011/0256180, the disclosure of which is incorporated by reference herein in its entirety and for
  • JAR 41 and JAR 64 mAbs bound equally well to fHbp variants, including representative variants in variant groups 1, 2 or 3 described by
  • Recombinant fHbps The gene encoding for each of nineteen fHbps (ID 1, ID 4,
  • ID 6 ID 9, ID 13, ID 14, ID 15, ID 19, ID 22, ID 28, ID 45, ID 55, ID 67, ID 74, ID 77, ID 79, ID 175, ID 207, and ID 283 from http://pubmlst.org/neisseria/fhbp) individually was cloned into an expression plasmid (pET21b) and expressed in Escherichia coli strain BL21(DE 3 ) as described in Masignani et al. (2003) J. Exp Med. 197:789-99.
  • the fHbp ID 1 R41S mutant that did not bind human fH was produced by site-directed mutagenesis as described in Beernink et al. (2011) J. Immunol.
  • Splenocytes were fused to mouse myeloma cell line P3X63-Ag8.653 as described in Welsch et al. ((2004) J. Immunol. 172:5606-15), and plated into 96-well microtiter plates. After two weeks of incubation, cell culture supernatants were collected and screened for production of antibody reactive to fHbp ID 1 by ELISA. Cell lines producing fHbp-reactive antibody were expanded and cloned by limiting dilution. Cloned cell lines were then re-screened by ELISA for continued production of antibody reactive to fHbp ID 1 by ELISA. This cloning process was repeated two to four times for each cell line to ensure monoclonality.
  • variable region gene sequences that encode antigen binding sites of the monoclonal antibodies were amplified by PCR from cDNA prepared from RNA that was isolated from hybridoma cells that secreted either JAR 41 or JAR 64, using degenerate primer mixes for variable light and variable heavy chains, as described in Wang et al. (2000) J Immunol Methods 233: 167-177. PCR products were then cloned into a sequencing vector and sequences were determined using a commercial service (Davis
  • the vector pYDl and the host yeast strain EBY100 were purchased from Invitrogen (Carlsbad, CA). Randomly mutated libraries of fHbp ID 1 were generated by error-prone PCR. MnCl 2 concentration were titrated, and conditions selected such that the average number of amino acid substitutions per molecule ranged from 1 to 3. These fragments were ligated into pUC18, expanded in E. coli, excised, and inserted into pYDl. Yeast were transformed with the plasmid library, expanded overnight at 30°C, transferred into Galactose-containing YNB medium (Yeast Nitrogen Base without amino acids (Difco) to induce recombinant protein expression, and incubated for 48 hrs at 20°C. Bulk yeast cultures were stained with both JAR 41 and JAR 3 antibodies, and sorted to select those yeast clones that had lost the ability to bind JAR 41 but retained binding to JAR 3 (this verifies surface expression of the construct).
  • Test antibody or a mouse immunoglobulin standard then were diluted serially into wells of sensitized and blocked microtiter plates and incubated overnight at 4°C. Plates were washed the following morning, and diluted alkaline phosphatase-conjugated goat antibody to mouse immunoglobulin light chain (Jackson ImmunoResearch) was added to the wells. After 1 hour incubation, the plates were washed again and phosphatase substrate was added to the wells. The optical density (OD 405 ) was measured after 30 minutes of incubation at room temperature. The concentration of antibody samples was assigned based on the OD 405 measurements of the dilutions of the mouse immunoglobulin standard.
  • the plates were washed and incubated for 1 hour at room temperature with an alkaline phosphatase- conjugated secondary goat antibody to mouse immunoglobulin. The plates were then washed and phosphatase substrate was added to wells. The OD 405 was measured after 30 minutes of incubation at room temperature.
  • JAR 41 Inhibition of binding of alkaline phosphatase-conjugated JAR 41 (AP-JAR 41) to fHbp by second anti-fHbp mAbs.
  • JAR 41 was purified using a protein G column (HiTrap Protein G, GE Lifesciences).
  • the mAb was conjugated to alkaline phosphatase (AP) using a commercial kit (Abeam, Cambridge, MA).
  • Percent inhibition was calculated using the formula: ((Average OD 405 of wells without inhibitor - OD 405 of sample well with inhibitor) / Average OD 405 of wells without inhibitor) x 100.
  • JAR 41 IgGl
  • JAR 64 IgGl
  • JAR 41 and JAR 64 mAbs tested as inhibitors of JAR 4 were of a different isotype not recognized by the anti-mouse IgG2a.
  • strain H44/76-SL B: 15:P1.7,16; sequence type 32 [ST-32]
  • H44/76 which expressed fHbp ID 1 (variant 1); strain 8047 (B:2b:P1.5-l, 2-2, sequence type 8 [ST-8]), which expressed fHbp ID 77 (variant 2,); and strain M1239 which expressed fHbp ID 28 (variant 3).
  • Binding to live N. meningitidis cells by flow cytometry Binding of monoclonal antibodies to the surface of live N. meningitidis was measured by flow cytometry as described in Welsch et al. (2004) J. Immunol. 172:5606-15. In brief, log-phase broth grown bacteria were distributed to 1.5 milliliter tubes, centrifuged, and washed in PBS buffer containing 1% bovine serum albumin (BSA). The bacteria were incubated with different concentrations of monoclonal antibody. Antibody bound to bacteria were detected by flow cytometry using a fluorescein isothiocyanate (FITC)-conjugated goat antibody to mouse immunoglobulin.
  • FITC fluorescein isothiocyanate
  • Bactericidal assay monoclonal antibodies were tested at different concentrations alone and in cooperation with a second monoclonal antibody raised against fHbp, as described in Beernink et al. ((2008) Infect. Immun. 76:4232-40). The assay used log-phase bacteria grown in 0.25% glucose and 0.02 mM cytidine monophospho-n-acetyl-neuraminic acid. The source of complement was a non-immune adult human serum that had been depleted of immunoglobulin G by passage through a protein G column (Hitrap Protein G, GE Healthcare), performed as described in Beernink et al. ((2011) J Immunol. 186:3606). The bactericidal 50% concentration for each antibody or combination of antibodies was defined as the total concentration of antibody resulting in a 50% decrease in colony forming units (CFU) after 60 minutes of incubation as compared with the CFU at time 0.
  • CFU colony forming units
  • JAR 41 and JAR 64 mAbs towards different fHbp variants were assayed by ELISA. These variants were selected as they are representative of all known fHbp modular and variant groups. Both mAbs showed broad reactivity with all 19 fHbp variants tested.
  • the concentration-dependent binding of JAR 41 mAb and JAR 64 mAB to the different fHbp variants is shown in Figures 3 and 4, respectively.
  • the respective concentration-dependent binding of JAR 41 mAb to the different fHbp variants was indistinguishable from that of JAR 64 mAb to the different fHbp variants.
  • control anti-fHbp mAbs showed the expected variant group specific binding, which matched that of the vaccine used for immunization of mice from which the control anti-fHbp mAbs were generated (see Table 2).
  • amino acid sequence variant as designated on the public data base, found at the website
  • JAR 5 (Welsch JA, et al. J Immunol 2004;172:5606-15; Beernink PT, et al. Infect Immun 2008;76:4232-4240) and mAb502 (Scarselli M, et al. J Mol Biol 2009; 386: 97-108) are control mAbs from wild type mice immunized with fHbp ID 1 (variant group 1).
  • JAR 63 is a
  • JAR 5, mAb502, and JAR 63 recognize a subset of fHbp sequence variants in variant group 1.
  • JAR 11 was from a mouse immunized with fHbp ID 16
  • JAR 35 was from a mouse immunized with fHbp ID 28 (variant group 3).
  • These mAbs cross-react with subsets of amino acid sequence variants in variant groups 2 and 3 but not variant group 1 (Beernink et al, Infection and Immunity Sep 2008;76:4232-4240).
  • Figure 5 illustrates the fHbp amino acid sequence variants recognized by JAR 41 and JAR 64.
  • Each filled circle represents a recombinant fHbp amino acid sequence variant that was bound by JAR 41 and JAR 64 in an ELISA ( Figures 3 and 4, respectively).
  • the individual sequence variants are designated by fHbp identification (ID) numbers as described on the public website.
  • fHbp ID 283 (a newly designated modular group X)
  • fHbp ID 207 (modular group VII) are natural hybrids with amino acid sequences that do not cluster within an individual sub-family or variant group.
  • Figure 6 depicts the inhibition of binding of alkaline phosphatase- conjugated anti-fHbp mAb JAR 41 to fHbp by JAR 41, JAR 64, JAR 4 or JAR 5. Concentrations of the inhibiting anti-fHbp mAb are shown on the X-axis. A 1: 10,000 dilution of alkaline phosphatase-conjugated JAR 41 gave an OD 40 5 of approximately 2.8 in the absence of an inhibitor. JAR 5, which binds avidly to fHbp ID 1, served as a negative control ( ⁇ 10
  • FIG. 6 (right panel) depicts inhibition of binding of JAR 4 (IgG2a) to fHbp by JAR 41 (IgGl), JAR 64 (IgGl) or JAR 5 (IgG2b).
  • a secondary alkaline phosphatase-conjugated anti-IgG antibody specific for mouse IgG2a was used to detect binding of JAR 4.
  • Both JAR 41 and JAR 64 inhibited binding of JAR 4 to fHbp.
  • JAR 4 epitope was shown to be discontinuous, involving DHK residues beginning at position 25 of fHbp ID 1; and YGN residues beginning at position 57 (Beernink et al. (2009) Molecular Micro . 46 (2009) 1647-1653.
  • the inhibition data in Figure 6 indicate that JAR 41 and JAR 64 recognize a similar region of the N-terminal domain of fHbp as did JAR 4.
  • Figure 7 illustrates binding of anti-fHbp mAbs to the surface of live bacteria as measured by flow cytometry.
  • Panel A JAR 41.
  • Panel B JAR 64.
  • the wildtype strains H44/76 (fHbp ID 1, variant group 1), 8047 (fHbp ID 77, variant 2 group), and M1239 (fHbp ID 28, variant 3 group), and the respective fHbp knock out (KO) mutants were tested.
  • the wild-type (WT) strains were incubated with 50 ⁇ g/ml (solid line) or 5 ⁇ g/ml (shaded area) of JAR 41 or JAR 64; the respective KO mutants (dashed lines) were incubated with 50 ⁇ g/ml of JAR 41 or JAR 64.
  • JAR 41 binds to the surface of live bacteria from group B strains H44/76 (fHbp ID 1 in variant group 1), 8047 (fHbp ID 77 in variant group 2), and M1239 (fHbp ID 28 in variant group 3) ( Figure 7, Panel A).
  • JAR 64 binds to strain H44/76 but not to 8047 or M1239 ( Figure 7, Panel B).
  • the difference in the ability of JAR 41 and JAR 64 to bind different meningococcal strains that express fHbps variants suggested that the two mAbs may recognize different but overlapping epitopes.
  • Figure 8 shows that anti-fHbp mAb JAR 41 is bactericidal with human complement when tested with second anti-fHbp mAbs against group B strain H44/76 (fHbp ID 1, variant group 1). Bactericidal activity of individual or combination of mouse anti-fHbp mAbs was tested with human complement. Error bars represent ranges in results from two independent assays. Control anti-PorA PI.7 or anti-capsular antibody (SEAM 12) mAbs were bactericidal individually. Individually, none of the anti-fHbp mAbs was bactericidal with human complement when tested at mAb concentrations up to 50 ⁇ g/ml.
  • JAR 41 + mAb502, or JAR 41 + JAR 5 were bactericidal against strain H44/76.
  • JAR 41 also was bactericidal against H44/76 in combination with a new anti-fHbp mAb, JAR 63, from a BALB/c human fH transgenic mouse immunized with a R41S mutant of fHbp ID 1 that does not bind human fH (See Table 2).
  • Figure 9 shows that anti-fHbp mAb JAR 41 is bactericidal with human complement when tested with second anti-fHbp mAbs against group B strain 8047 (fHbp ID 77, variant group 2). Bactericidal activity of individual or combination of mouse anti-fHbp mAbs was tested with human complement. Control anti-PorA PI.2 or anticapsular antibody (SEAM 12) mAbs were bactericidal individually. Individually, none of the anti-fHbp mAbs was bactericidal with human complement when tested at mAb concentrations up to 100 ⁇ g/ml.
  • JAR 41 + JAR 11 or JAR 41 + JAR 13 were bactericidal against strain 8047.
  • the bactericidal activity of the combinations of JAR 41 with JAR 11 or JAR 13 against strain 8047 was confirmed in a second experiment.
  • JAR 41 was not bactericidal, individually or in combination, when tested with human complement against group B strain M1239.
  • JAR 64 lacked human complement-mediated bactericidal activity when tested individually or in combination with second anti-fHbp mAbs against H44/76.
  • FIG. 10A and 10B Variable heavy and light chain sequences of JAR 41 ( Figures 10A and 10B, respectively) and of JAR 64 ( Figures 11A and 11B) encoded different amino acid sequences.
  • Figure 12 shows the amino acid sequences of the JAR 41 and JAR 64 variable heavy and variable light chain CDRs aligned with the respective germline sequences. Alignment of JAR 41, JAR 64, and JAR 4 CDR regions. CDRs are as defined by Kabat (Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. 1991. Sequences of Proteins of Immunological Interest, 5 th ed, vol. 2. U.S. Department of Health and Human Services, Bethesda.).
  • Germline gene assignments were based on IgBLAST (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSTBLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402).
  • "Homology” in Figure 12 refers to percent nucleotide homology to the assigned germline gene over the V region. Shown for comparison are the corresponding sequences of a third anti-fHbp, JAR 4 (Welsch et al. (2004) J. Immunol. 172:5606; and Beernink et al. (2009) Mol. Immunol.
  • JAR 41 and JAR 64 variable heavy chain sequences match with different germline variable sequences (J558.35 and J558.2, respectively). Although the variable light chain sequences of JAR 41 and JAR 64 both match the 21.4 germline variable sequence, the light chain sequences of both mAbs are nevertheless different from one another. Thus JAR 41 and JAR 64 are distinctly different antibodies that recognize overlapping epitopes on fHbp.
  • the CDRs of the two mAbs also are different from those encoding the previously described mAb, JAR 4.
  • yeast expression system was used to screen a randomly mutated fHbp ID 1 library for expression of sequences encoding proteins that lacked JAR 41 binding. Yeast were sorted by flow cytometry for positive binding to JAR 3 (which served as a positive control for fHbp), but negative binding to JAR 41.
  • Figure 13 depicts representative flow cytometry histograms of yeast that express an empty vector (left panel),which show background binding with JAR 3 (dashed line) and JAR 41 (gray filled histogram).
  • Binding of both mAbs to yeast expressing wild-type fHbp ID 1 is shown in the center panel, and binding of yeast with JAR 3 only is shown in the right panel. Absence of binding of JAR 41 to the fHbp protein expressed by yeast in the right panel suggested that the amino acid sequence of the fHbp contained a mutation that was important for binding of JAR 41.
  • the D25A mutation resulted in complete loss of binding of both JAR 41 and JAR 64, and partial loss of JAR 4 binding ( Figure 14).
  • the K27A mutation resulted in complete loss of binding to JAR 4 but had no significant effect on binding of JAR 41 or JAR 64.
  • JAR 41 and JAR 64 bind to epitopes that overlap but are distinctive from that of the previously described JAR 4 epitope.
  • IP intraperitoneally
  • Control rats received buffer alone (phosphate buffered saline, PBS) or 12.5 ⁇ g of an anti-PorA mAb (PI.7).
  • Total volume of treatment for each animal was 100 ⁇ .
  • JAR 5 individually conferred incomplete protection (2 of 5 animals had sterile blood cultures). JAR 41 individually did not confer protection (all animals with bacteremia). The combination of JAR 5 + JAR 41 conferred complete protection (all five animals with sterile cultures; geometric mean CFU/ml ⁇ 5 compared with 70 in PBS treated animals; 95% confidence interval, 11 to 455).
  • JAR 41 individually is not bactericidal in vitro ( Figure 9), or protective in vivo (Figure 16), JAR41 augments bactericidal activity (See Figure 9), and protective activity ( Figure 16) of some second anti-fHbp mAbs such as JAR 5 against meningococcal bacteremia in the human transgenic infant rat model.
  • JAR 64 monoclonal antibodies were deposited under the terms of the Budapest Treaty with the American Type Culture Collection, 10801 University Boulevard., Manassas, Va. 20110-2209, USA (ATCC) on the date indicated in the table below, and were assigned the designations set out in Table 4, below.
  • JAR 5 mAb specifically binds to an epitope that at least partially overlaps with the epitope specifically bound by JAR 3 mAb
  • JAR 32 mAb specifically binds to an epitope that at least overlaps with the epitope specifically bounds by JAR 35 mAb.

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Abstract

La présente invention concerne des anticorps qui se lient spécifiquement à la protéine liant le facteur H. Les anticorps sont utiles dans diverses applications, qui sont également concernées par l'invention.
PCT/US2012/066099 2011-11-23 2012-11-20 Anticorps anti-protéine liant le facteur h et procédés pour les utiliser Ceased WO2013078223A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9439957B2 (en) 2010-03-30 2016-09-13 Children's Hospital & Research Center Oakland Factor H binding proteins (FHBP) with altered properties and methods of use thereof
US9914756B2 (en) 2013-08-02 2018-03-13 Children's Hospital & Research Center At Oakland Non-naturally occurring factor H binding proteins (fHbp) and methods of use thereof
US10266572B2 (en) 2014-07-23 2019-04-23 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
WO2021011903A1 (fr) * 2019-07-17 2021-01-21 Gemini Therapeutics Inc. Anticorps de potentialisation du facteur h et leurs utilisations
US12497432B2 (en) * 2016-08-31 2025-12-16 Oxford University Innovation Limited Modified factor H binding protein

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020065398A1 (en) * 1994-11-18 2002-05-30 Cristina M.M.A. Del Rio Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (egf-r); diagnostic and therapeutic use
WO2010037835A2 (fr) * 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique pscaxcd3 (cd19xcd3, c-metxcd3, endosialinxcd3, epcamxc d3, igf-1rxcd3 ou fapalpha xcd3), spécifique d'espèces croisées
US20100105875A1 (en) * 2008-06-09 2010-04-29 Maria Scarselli Antibodies against neisserial factor H binding protein
US20100266617A1 (en) * 2007-06-18 2010-10-21 N.V. Organon Antibodies to human programmed death receptor pd-1
WO2011004028A2 (fr) * 2009-07-10 2011-01-13 Innate Pharma Agents de liaison à tlr3
US20110189171A1 (en) * 2007-03-06 2011-08-04 Symphogen A/S Recombinant antibodies for treatment of respiratory syncytial virus infections
US20110200595A1 (en) * 2010-02-18 2011-08-18 Roche Glycart TREATMENT WITH A HUMANIZED IgG CLASS ANTI EGFR ANTIBODY AND AN ANTIBODY AGAINST INSULIN LIKE GROWTH FACTOR 1 RECEPTOR
US20110236377A1 (en) * 2006-06-02 2011-09-29 May Han Hepatocyte Growth Factor (HGF) Binding Proteins
US20110263436A1 (en) * 2006-12-05 2011-10-27 Abbott Laboratories Recombinant antibodies against hepatitis c virus and methods of obtaining and using same

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020065398A1 (en) * 1994-11-18 2002-05-30 Cristina M.M.A. Del Rio Humanized and chimeric monoclonal antibodies that recognize epidermal growth factor receptor (egf-r); diagnostic and therapeutic use
US20110236377A1 (en) * 2006-06-02 2011-09-29 May Han Hepatocyte Growth Factor (HGF) Binding Proteins
US20110263436A1 (en) * 2006-12-05 2011-10-27 Abbott Laboratories Recombinant antibodies against hepatitis c virus and methods of obtaining and using same
US20110189171A1 (en) * 2007-03-06 2011-08-04 Symphogen A/S Recombinant antibodies for treatment of respiratory syncytial virus infections
US20100266617A1 (en) * 2007-06-18 2010-10-21 N.V. Organon Antibodies to human programmed death receptor pd-1
US20100105875A1 (en) * 2008-06-09 2010-04-29 Maria Scarselli Antibodies against neisserial factor H binding protein
WO2010037835A2 (fr) * 2008-10-01 2010-04-08 Micromet Ag Anticorps monocaténaire bispécifique pscaxcd3 (cd19xcd3, c-metxcd3, endosialinxcd3, epcamxc d3, igf-1rxcd3 ou fapalpha xcd3), spécifique d'espèces croisées
WO2011004028A2 (fr) * 2009-07-10 2011-01-13 Innate Pharma Agents de liaison à tlr3
US20110200595A1 (en) * 2010-02-18 2011-08-18 Roche Glycart TREATMENT WITH A HUMANIZED IgG CLASS ANTI EGFR ANTIBODY AND AN ANTIBODY AGAINST INSULIN LIKE GROWTH FACTOR 1 RECEPTOR

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 19 June 2005 (2005-06-19), "immunoglobulin heavy chain [Mus musculus]", retrieved from http://www.ncbi.nlm.nih.gov/protein/AAX90201 accession no. AX90201 *
DATABASE GENBANK 23 November 2005 (2005-11-23), "Immunoglobulin heavy chain CDR3 [Homo sapiens].", retrieved from http://www.ncbi.nlm.nih.gov/protein/AAT02122 accession no. AT02122 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10905754B2 (en) 2010-03-30 2021-02-02 Children's Hospital & Research Center At Oakland Factor H binding proteins (fHbp) with altered properties and methods of use thereof
US9827300B2 (en) 2010-03-30 2017-11-28 Children's Hospital & Research Center Oakland Factor H binding proteins (FHBP) with altered properties and methods of use thereof
US9439957B2 (en) 2010-03-30 2016-09-13 Children's Hospital & Research Center Oakland Factor H binding proteins (FHBP) with altered properties and methods of use thereof
US10342860B2 (en) 2010-03-30 2019-07-09 Children's Hospital & Research Center At Oakland Factor H binding proteins (FHBP) with altered properties and methods of use thereof
US9914756B2 (en) 2013-08-02 2018-03-13 Children's Hospital & Research Center At Oakland Non-naturally occurring factor H binding proteins (fHbp) and methods of use thereof
US10995122B2 (en) 2014-07-23 2021-05-04 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US10487122B2 (en) 2014-07-23 2019-11-26 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US10836799B2 (en) 2014-07-23 2020-11-17 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US10266572B2 (en) 2014-07-23 2019-04-23 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US11673920B2 (en) 2014-07-23 2023-06-13 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US11834476B2 (en) 2014-07-23 2023-12-05 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US12129282B2 (en) 2014-07-23 2024-10-29 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US12269849B2 (en) 2014-07-23 2025-04-08 Children's Hospital & Research Center At Oakland Factor H binding protein variants and methods of use thereof
US12497432B2 (en) * 2016-08-31 2025-12-16 Oxford University Innovation Limited Modified factor H binding protein
WO2021011903A1 (fr) * 2019-07-17 2021-01-21 Gemini Therapeutics Inc. Anticorps de potentialisation du facteur h et leurs utilisations
US11820814B2 (en) 2019-07-17 2023-11-21 Gemini Therapeutics Sub, Inc. Factor H potentiating antibodies and uses thereof

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