WO2008116118A2 - Anticorps totalement humains de bactéries à gram positif - Google Patents

Anticorps totalement humains de bactéries à gram positif Download PDF

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Publication number
WO2008116118A2
WO2008116118A2 PCT/US2008/057750 US2008057750W WO2008116118A2 WO 2008116118 A2 WO2008116118 A2 WO 2008116118A2 US 2008057750 W US2008057750 W US 2008057750W WO 2008116118 A2 WO2008116118 A2 WO 2008116118A2
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seq
antibody
fully human
nos
amino acid
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PCT/US2008/057750
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English (en)
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WO2008116118A3 (fr
Inventor
Ritsuko Sawada
Wolfgang W. Scholz
Paul Ruther
Ivy Jiang
Shu-Man Sun
Fei Wang
Angray Kang
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Avanir Pharmaceuticals
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Publication of WO2008116118A3 publication Critical patent/WO2008116118A3/fr

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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
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • This invention in the fields of immunology and infectious diseases relates generally to fully human monoclonal antibodies that mediate opsonophagocytic killing of Staphylococcus aureus (S. aureus) and other Gram positive bacteria.
  • Embodiments of the invention include human monoclonal antibodies or fragments thereof, which may be used for diagnostic, prophylactic and therapeutic treatment applications.
  • Staphylococcus aureus and coagulase-negative staphylococci are among the most common causes of nosocomial infections in the intensive care unit (ICU).
  • S. aureus infections result in a broad spectrum of diseases including skin infections, endocarditis, arthritis, osteomyelitis, sepsis, device-related infections, pneumonias, and surgical wound infections (Bassetti, S., S. Wasmer, et al. (2005). "Staphylococcus aureus in patients with rheumatoid arthritis under conventional and anti-tumor necrosis factor- alpha treatment.” J. Rheumatol. 32(11): 2125-9.).
  • Oxacillin resistant S. aureus (MRSA) infections are observed primarily in hospital settings, but reports of community acquired MRSA infections (CA- MRSA) are alarmingly increasing. Moreover, several examples exist where S. aureus infections could not be cleared by vancomycin, which is considered the last resort for effective antibiotic treatment.
  • S. aureus is a leading cause of hospital-based blood stream infections and has a crude mortality of 25 percent.
  • S. epidermidis is a major cause of nosocomial infections, including sepsis in premature infants (Fischer, G. W., T. J. Cieslak, et al. (1994). "Opsonic antibodies to Staphylococcus epidermidis: in vitro and in vivo studies using human intravenous immune globulin.” J. Infect. Dis. 169(2): 324-9.).
  • New therapeutics designed to address the medical need that are currently in clinical development include a staphylococcus vaccine (StaphVaxTM, Nabi), as well as a humanized monoclonal antibodies against lipoteichoic acid (LTA) (US Patent Nos. 6,610,293 Bl, 6,939,543 B2, and 7,250,494 B2), and a humanized antibody against clumping factor A (Domanski, P. J., P. R. Patel, et al. (2005). "Characterization of a Humanized Monoclonal Antibody Recognizing Clumping Factor A Expressed by Staphylococcus aureus.” Infect. Immun. 73(8): 5229-5232.).
  • Other antibodies have also been disclosed (U.S. Patent No. 6,692,739 and U.S. Patent Publication No. 2004/0006209).
  • Lipoteichoic acid found in Gram positive bacteria has been characterized extensively (Wicken, A. J. and K. W. Knox (1975). "Lipoteichoic acids: a new class of bacterial antigen.” Science 187(4182): 1161-7; Martin, R. R., S. B. Greenberg, et al. (1979). "Staphylococcal teichoic-acid antibodies.” Lancet 1(8118): 731; Raynor, R. H., D. F. Scott, et al. (1981). "Lipoteichoic acid inhibition of phagocytosis of Staphylococcus aureus by human polymorphonuclear leukocytes.” Clin. Immunol.
  • Anti- S. aureus antibodies which recognize bacterial cell surface antigens are thought to provide protection against S. aureus infections by the rapid clearance of the organism through opsonophagocytosis (Verbrugh, H. A., W. C. Van Dijk, et al. (1979). "Staphylococcus aureus opsonization mediated via the classical and alternative complement pathways. A kinetic study using MgEGTA chelated serum and human sera deficient in IgG and complement factors CIs and C2.” Immunology 36(3): 391-7; Weisman, L. E., D. F. Cruess, et al. (1994). "Opsonic activity of commercially available standard intravenous immunoglobulin preparations.” Pediatr. Infect. Dis. J. 13(12): 1122- 5.). However, the selection of the most appropriate development candidate among anti- LTA antibodies is not straightforward.
  • inventions of the invention comprise fully human antibodies, and fragments thereof, that bind to Staphylococcus bacteria and/or bacterial components or products (such as LTA).
  • the invention comprises the generation and selection of fully human anti-LTA antibodies.
  • human antibodies are obtained from naturally exposed human subjects and selected by measuring the binding to multiple strains of S. aureus and the in vitro opsonophagocytic killing activity mediated by these human antibodies.
  • Several embodiments of the present invention comprise four distinct human monoclonal antibodies that mediate opsonisation and phagocytosis of S. aureus whole bacteria and/or other Staphylococcus species.
  • these antibodies bind to LTA that is expressed on the surface of the bacteria.
  • These antibodies are selected based on their ability to enhance the killing of bacteria by human effector cells, such as polymorphonuclear neutrophils (PMN) in vitro, and to provide protection from lethal infection in vivo.
  • PMN polymorphonuclear neutrophils
  • These antibodies are molecularly cloned and expressed as recombinant proteins in CHO cells.
  • the invention comprises a cell (e.g., a modified cell or CHO cell) comprising one or more the antibodies described herein.
  • the invention comprises a fully human monoclonal antibody, wherein the antibody or fragment thereof is encoded at least in part by an amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
  • the invention comprises a hybridoma comprising one or more of the polynucleotides described herein.
  • the invention comprises a fully human antibody, wherein the antibody or fragment thereof is encoded at least in part by amino acid sequences selected from the group consisting of one or more of the following pairs of sequences: SEQ ID NO: 1 + SEQ ID NO: 2, SEQ ID NO: 3 + SEQ ID NO: 4, SEQ ID NO: 5 + SEQ ID NO: 6, and SEQ ID NO: 7 + SEQ ID NO: 8.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody recognizes infectious bacteria.
  • the bacteria comprise Gram positive bacteria.
  • the bacteria comprise S. aureus (including antibiotic resistant strains of S. aureus, such as methicillin-resistant S. aureus).
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody recognizes one or more of the following bacterial strains: S. warneri, S. aureus, S. epidermidis, S. haemofyticus, S. hominis, and S. saprophyticus.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody recognizes the LTA portion of a bacterial strain or isolated LTA.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody has substantial opsonophagocytic killing activity in vitro when tested with human effector cells and human complement.
  • the killing activity is greater than about 50%.
  • the killing activity is about 75%.
  • the killing activity is about 100%.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody will have killing activity in vivo.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody is used in the prophylaxis or treatment of one or more of the following pathologies: skin abscesses, septic arthritis, osteomyelitis, infective endocarditis, surgical wound infections, and infections associated with medical devices.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody is used in the prophylaxis or treatment of one or more of the following pathologies: MRSA, blood stream infections, and S. epidermidis infections.
  • the invention comprises a fully human antibody according to any one of the embodiments described herein, wherein the antibody is used in the prophylaxis or treatment in one or more of the following patients: low birth weight infants, immuno-compromised individuals, hemodialysis patients, and individuals undergoing elective surgery.
  • the antibody is used in the prophylaxis or treatment in one or more of the following patients: low birth weight infants, immuno-compromised individuals, hemodialysis patients, and individuals undergoing elective surgery.
  • patients low birth weight infants, immuno-compromised individuals, hemodialysis patients, and individuals undergoing elective surgery.
  • the invention comprises a pharmaceutical formulation comprising a fully human antibody according to any one of the embodiments described herein.
  • the pharmaceutical formulation comprises an additional therapeutic agent.
  • the additional therapeutic agent is operable to treat bacterial infections and/or viral infections.
  • the additional therapeutic agent includes, but is not limited to, one or more of the following compounds: a second antibody (human, non-human, or humanized), a vaccine, an antibiotic, an antiviral, and a non-antibiotic anti-infective compound.
  • the additional therapeutic agents can be administered simultaneously or sequentially with said one or more fully human monoclonal antibodies.
  • the invention comprises a diagnostic or therapeutic kit comprising a fully human antibody or formulation according to any one of the embodiments described herein.
  • the kit comprises at least one means to administer the antibody or formulation.
  • the kit comprises instructions for use.
  • the invention comprises a method of treating infections caused or mediated by bacteria (such as S. aureus and/or other Staphylococcus species), wherein the method comprises administering a therapeutic dose of a fully human antibody according to any one of the embodiments described herein.
  • bacteria such as S. aureus and/or other Staphylococcus species
  • the invention comprises a method for passively immunizing a mammal against bacterial infection (such as that caused by S. aureus and/or other Staphylococcus species), comprising administering an immunizing dose of a fully human antibody according to any one of the embodiments described herein.
  • the antibody or formulation is administered parenterally, topically, or orally.
  • a medical device such as a syringe, patch, or inhaler is used for administration.
  • the invention comprises a pharmaceutical formulation comprising an antibody according to any of the embodiments described herein and at least one other additional therapeutic agent.
  • a method of preventing or treating infection or illness comprises administering the pharmaceutical formulation.
  • the additional therapeutic agent can be administered simultaneously or sequentially.
  • the invention comprises a fully human monoclonal antibody that recognizes lipoteichoic acid
  • the fully human monoclonal antibody is made by a method comprising: (1) administering peripheral blood cells from one or more human donors exposed to lipoteichoic acid to a SCID mouse (or other immunocompromised animal), (2) administering one or more doses of lipoteichoic acid to the mouse, (3) isolating at least one immune cell ⁇ e.g., lymphocytic cell) from the mouse, and (4) fusing the immune cell (e.g., lymphocytic cell) with a fusion partner, thereby generating a hybridoma, wherein the hybridoma produces a fully human monoclonal antibody which recognizes lipoteichoic acid.
  • a SCID mouse or other immunocompromised animal
  • the immune cell e.g., lymphocytic cell
  • the immune cell may be cultured or immortalized using other techniques known in the art (e.g., culturing spontaneous transformed cells in the SCID mouse, immortalization with a suitable oncogene, etc.), wherein the derived cells produce a fully human monoclonal antibody which recognizes lipoteichoic acid.
  • the invention comprises a fully human monoclonal antibody that recognizes lipoteichoic acid, wherein the fully human monoclonal antibody is made by a method comprising: (a) obtaining human blood from individuals exposed to infectious bacteria; (b) selecting Human B cells (e.g., by magnetic beads); (c) transforming B cells by Epstein Barr Virus (EBV); (d) culturing B cells (e.g., on a feeder layer); testing for antibodies; (e) harvesting cells; and (f) isolating RNA to make recombinant antibody. Methods of using RNA to make recombinant antibody, are well known in the art.
  • the invention comprises a fully human monoclonal antibody that recognizes lipoteichoic acid, wherein said antibody is obtainable by a process comprising: (a) obtaining human blood from one or more humans exposed to bacteria, wherein the bacteria comprise lipoteichoic acid; (b) selecting B cells from the blood; (c) transforming said B cells using EBV; (d) culturing said B cells; (e) testing said culture for the production of antibodies that bind to lipoteichoic acid; (f) harvesting cells that produce antibodies that bind to lipoteichoic acid; (g) isolating nucleic acid from said antibody-producing cells; and (h) using said isolated nucleic acid to make a recombinant fully human monoclonal antibody that recognizes lipoteichoic acid.
  • Methods of using nucleic acid to make recombinant antibody are well known in the art.
  • the invention comprises an isolated polypeptide selected from one or more of the following: a protein with a complete amino acid sequence encoded in any of SEQ ID NOs: 1-8; a fragment of said protein; and a fusion protein containing said protein or said fragment, wherein the isolated polypeptide binds to lipoteichoic acid.
  • Nucleotide sequences corresponding to SEQ ID NOs: 1-8 are provided as SEQ ID NOs: 33-40, respectively.
  • the invention comprises a method for preventing or treating a bacterial infection in a patient caused by bacteria comprising lipoteichoic acid, wherein the method comprises administering to the patient an isolated protein comprising: a complete amino acid sequence encoded in any of SEQ ID NOs: 1-8; or an amino acid sequence that is at least about 80% identical to the amino acid sequences of any of SEQ ID NOs: 1-8, wherein the isolated protein binds to lipoteichoic acid, thereby preventing or treating said bacterial infection.
  • identity is about 95% or 99%.
  • the invention comprises an isolated protein comprising ⁇ e.g., encoded by) an amino acid sequence that is at about least 80%-95% identical to any of the amino acid sequences shown in SEQ ID NOs: 1-8 and binds to lipoteichoic acid.
  • the invention comprises an isolated protein comprising an amino acid sequence that is identical to any of the CDR amino acid sequences shown in SEQ ID NOs: 1-8 and binds to lipoteichoic acid.
  • the invention comprises an isolated protein comprising an amino acid sequence that is about 80-95% identical to any of the CDR amino acid sequences shown in SEQ ID NOs: 1-8 and binds to lipoteichoic acid.
  • the invention comprises an amino acid sequence that is partially homologous ⁇ e.g., about 80%, 85%, 90%, 95%, or 99% identical) to any of the CDR amino acid sequences shown in SEQ ID NOs: 1-8.
  • variants of the nucleic acid or amino acid sequences listed herein comprise variants of the nucleic acid or amino acid sequences listed herein.
  • one or several of the amino acid residues in the variable heavy and/or light chain sequences are modified by substitution, addition, and/or deletion such that binding to LTA is not substantially affected.
  • the present invention comprises variants that contain conservative substitution mutations.
  • conservative substitution refers to the substitution of an amino acid within the same general class ⁇ e.g., an acidic amino acid, or a basic amino acid, a neutral amino acid) by another amino acid within the same class.
  • the invention comprises one or more of the amino acid sequences shown in SEQ ID NOs: 1-8, except that at least one of the amino acids listed is exchanged for an amino acid in the same class.
  • the invention comprises one or more of the amino acid sequences shown in SEQ ID NOs: 1-8, except that at least one of the amino acids listed is exchanged for an amino acid in a different class.
  • nucleotide sequence provided in any of SEQ ID NOs: 33-64 comprising at least one silent nucleotide substitution.
  • the variant will have at least about 95% of the binding affinity as any one of AVP-6B10, V-7-27, 3E7 and 15C8S. In another embodiment, the variant will have at least about 75% of the in vitro functional activity of an antibody encoded by any one of AVP-6B10, V-7-27, 3E7 and 15C8S.
  • the invention comprises an isolated amino acid sequence or an isolated protein encoded by an amino acid sequence selected from one or more of the following: SEQ ID NOs: 9-32. Nucleotide sequences corresponding to SEQ ID NOs: 9-32 are provided as SEQ ID NOs: 41-64.
  • the invention comprises a fully human antibody that recognizes LTA and comprises one of the following four CDR amino acid sequence sets:
  • SEQ ID NO: 9 (15C8S, CDRlH): GFTLSAYWMT
  • SEQ ID NO: 10 (15C8S, CDR2H): NIKGDGSKEYYVDSVKG
  • SEQ ID NO: 11 (15C8S, CDR3H): DSNPQPSGNHYYDAFDV
  • SEQ ID NO: 12 (15C8S, CDRlL): RTSQGIRDDLG
  • SEQ ID NO: 15 (6B10, CDRlH): WSTFSGSAMH
  • SEQ ID NO: 16 (6B10, CDR2H):YIRSKTKNYATSYAASVRG
  • SEQ ID NO: 17 (6B10, CDR3H): HAEFDRSH
  • SEQ ID NO: 18 (6B10, CDRlL): RASQDISN
  • SEQ ID NO: 20 (6B10, CDR3L): QQYRSSPWT
  • SEQ ID NO: 21 (3E7, CDRlH): GFTFNTFAMN
  • SEQ ID NO 22 (3E7, CDR2H): GISGSGETTYYADSVKG
  • SEQ ID NO: 23 (3E7, CDR3H): RPGIKAGGGDH
  • SEQ ID NO: 24 (3E7, CDRlL): RSSQSLLNYNGNYYLD
  • SEQ ID NO: 25 (3E7, CDR2L): LSSRRAS
  • SEQ ID NO: 27 (V-7-27, CDRlH): GFIFNTNAMS
  • SEQ ID NO: 28 (V-7-27, CDR2H): GINARGATIYYADSVKG
  • SEQ ID NO: 30 (V-7-27, CDRlL): TGTTSDVGSYSFVS
  • SEQ ID NO: 32 (V-7-27, CDR3L): CSYAGNSIYV
  • the invention comprises a fully human antibody that recognizes LTA and comprises one of the following eight CDR amino acid sequence sets:
  • SEQ ID NO: 9 (15C8S, CDRlH): GFTLSAYWMT
  • SEQ ID NO: 10 (15C8S, CDR2H): NIKGDGSKEYYVDSVKG
  • SEQ ID NO: 11 (15C8S, CDR3H): DSNPQPSGNHYYDAFDV
  • SEQ ID NO: 12 (15C8S, CDRlL): RTSQGIRDDLG
  • SEQ ID NO: 15 (6B10, CDRlH): WSTFSGSAMH
  • SEQ ID NO: 16 (6B10, CDR2H):YIRSKTKNYATS YAASVRG
  • SEQ ID NO: 17 (6B10, CDR3H): HAEFDRSH
  • SEQ ID NO: 18 (6B10, CDRlL): RASQDISN
  • SEQ ID NO: 20 (6B10, CDR3L): QQYRSSPWT
  • SEQ ID NO: 21 (3E7, CDRlH): GFTFNTFAMN
  • SEQ ID NO 22 (3E7, CDR2H): GISGSGETTYYADSVKG
  • SEQ ID NO: 23 (3E7, CDR3H): RPGIKAGGGDH
  • SEQ ID NO: 25 (3E7, CDR2L): LSSRRAS
  • SEQ ID NO: 27 (V-7-27, CDRlH): GFIFNTNAMS
  • SEQ ID NO: 28 (V-7-27, CDR2H): GIN ARG ATI Y YAD SVKG
  • SEQ ID NO: 30 (V-7-27, CDRlL): TGTTSDVGS YSFVS
  • SEQ ID NO: 32 (V-7-27, CDR3L): CSYAGNSIYV
  • the invention comprises an antibody or protein according to any of the embodiments described herein, wherein the antibody or protein is cloned and expressed as a recombinant protein in Chinese hamster ovary (CHO) cells.
  • CHO Chinese hamster ovary
  • NSO cells or other mammalian cells, plant cells, or bacteria are used instead of CHO cells.
  • the invention comprises a cell line expressing a fully human antibody according to any of the embodiments described herein.
  • the cell line is a stable CHO cell line.
  • the invention comprises a method of expressing antibodies or proteins having binding activity to LTA, comprising: subcloning heavy or light variable cDNA regions encoding of any one of SEQ ID NOs: 1-8 into expressions cassette and co-expressing said cDNA regions in CHO cells.
  • a dual expression vector is constructed by ligating the heavy and light chain into a single expression cassette, thereby establishing individual anti-Staphylococcus antibodies.
  • the invention comprises an antibody comprising one or more of the nucleotide sequences or amino acid sequences described herein.
  • the invention comprises one or more of the nucleotide sequences or amino acid sequences described herein - in other words, in some embodiments, the nucleotide sequence or amino acid sequences need not be expressed into a polypeptide or antibody.
  • the sequences themselves have several utilities, including uses in screening and diagnosis.
  • the invention comprises a method of screening a sample for bacteria (such as S.
  • aureus comprising contacting at least a portion of the sample with a fully human antibody according to any one of the preceding claims, determining the presence or absence of binding of the antibody to the bacteria (e.g., S. aureus), wherein the presence of binding indicates that the sample contains bacteria (e.g., S. aureus), and wherein the absence of binding indicates that the sample is substantially free of bacteria (e.g., S. aureus).
  • This method may be particularly useful in hospitals in determining whether or not a certain patient is infected. Alternatively, this method may be useful in determining whether certain hospital rooms, clothing, surfaces etc are contaminated with bacteria.
  • Some embodiments provide a fully human monoclonal antibody, wherein said antibody or fragment thereof is encoded at least in part by polynucleotide comprising a nucleotide sequence selected from the group consisting of one or more of the following: SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40.
  • Some embodiments provide fully human antibody, wherein said antibody or fragment thereof is encoded at least in part by a cDNA sequence selected from the group consisting of one or more of the following: SEQ ID NO: 33 + SEQ ID NO: 34, SEQ ID NO: 35 + SEQ ID NO: 36, SEQ ID NO: 37 + SEQ ID NO: 38, and SEQ ID NO: 39 + SEQ ID NO: 40.
  • Some embodiments provide fully human antibody, wherein said antibody or fragment thereof is encoded at least in part by a polynucleotide comprising nucleotide sequences selected from the group consisting of one or more of the four following groups of sequences: SEQ ID NOs: 41-46; SEQ ID NOs: 47-52; SEQ ID NOs: 53-58; and SEQ ID NOs: 59-64.
  • Some embodiments provide fully human antibody, wherein said antibody or fragment thereof is encoded at least in part by a polynucleotide comprising nucleotide sequences selected from the group consisting of one or more of the eight following groups of sequences: SEQ ID NOs: 41-43; SEQ ID NOs: 44 ⁇ 16; SEQ ID NOs: 47 ⁇ 19; SEQ ID NOs: 50-52; SEQ ID NOs: 53-55; SEQ ID NOs: 56-58; SEQ ID NOs: 59-61, and SEQ ID NOs: 62-64. [0047] Some embodiments provide fully human antibody, wherein said antibody or fragment thereof is encoded at least in part by at least one nucleotide sequence identified in any one of SEQ ID NOs: 41-64.
  • FIG. 1 shows serum samples from 20 normal blood donors tested for binding to S. aureus whole bacteria (Type 8) by ELISA (A). Presence of functional antibodies in these samples was measured in an opsonophagocytic killing assay (B).
  • FIG. 2 shows the binding of selected human mAbs to S. aureus and LTA from various bacteria as detected in an ELISA assay.
  • A Binding to S. aureus type 5, type 8 and Woods strain as well as S. aureus derived LTA.
  • B Binding of human mAbs to LTA isolated from S. aureus (two different batches), three different strains of Streptococcus, or Bacillus subtilis.
  • C Binding of human mAbs to Staphylococcus epidermidis.
  • FIG. 3 shows detection of the binding of 6B10 and V-7-27 human mAbs to S. aureus by immunofluorescence.
  • Bacteria treated with secondary antibody alone or a control antibody (IgG/ ⁇ ), which recognizes an unrelated antigen (protective antigen from Bacillus anthracis) do not show fluorescent staining.
  • FIG. 4 shows dose-dependent opsonophagocytic killing of S. aureus type 8 by human PMN in presence of AVP-6B10, V-7-27, 3E7, and 15C8S (A).
  • Bacterial killing activity of human IgGj (/Gl or no extra label) and human IgG 3 (/G3) isotypes of AVP-6B10, V-7-27, 3E7, and 15C8S against Staphylococcus epidermidis is shown in (B).
  • FIG. 5 shows opsonophagocytic killing of S. aureus type 5 (A) or MRSA strain BAA-41 (B) by human PMN in presence of AVP-6B10, V-7-27, 3E7 and 15C8S.
  • AVP-6B10, 3E7, and V-7-27 were expressed and tested either in the Gi (-/Gl) or the G 3 isoform ((-/G3) of human immunoglobulin.
  • Human AVP-21D9 antibodies that recognize a non-related antigen served as negative control (A).
  • FIG. 6 shows that treatment with AVP-3E7/G3 protects mice challenged with a lethal dose of S. aureus BAA-41.
  • FIG. 7 shows that treatment with AVP-V-7-27 protects mice challenged with a lethal dose of S. aureus type 5.
  • FIG. 8 shows Fc receptor binding of human mAbs to murine macrophages and PMN (A) or rat PMN (B).
  • Human mAbs of IgG 3 isotype bind significantly better to both rodent Fc receptors compared to antibodies containing the human IgGi isotype.
  • FIG. 9 shows the variable coding region of AVP-15C8S.
  • FIG. 10 shows the variable coding region of AVP-6B10.
  • FIG. 1 1 shows the variable coding region of AVP-3E7.
  • FIG. 12 shows the variable coding region of AVP-V-7-27.
  • FIG. 13 shows TABLE 1, which shows antigens (LTA, whole bacteria, clumping factor A) that are used for generation of human monoclonal antibodies against S. aureus. Antibodies that are generated are further selected for functional activity in an opsonophagocytic killing assay.
  • FIG. 14 shows TABLE 2, which shows examples of human mAbs generated by immunization with LTA, whole bacteria or recombinant clumping factor A and the binding of the antibodies to whole bacteria (3 different strains) in an ELISA assay.
  • FIG. 15 shows TABLE 3, which summarizes the results obtained for six different human anti S. aureus antibodies in OPKA assays utilizing three different strains of S. aureus. All assays were performed with human PMN and human complement.
  • FIG. 16 shows TABLE 4, which shows that isotype switching to a different human immunoglobulin class does not impair the binding of the antibodies to whole S. aureus bacteria.
  • FIG. 17 shows TABLE 5, which shows the bacteria that were purchased from ATTC and used in the studies described in this invention.
  • FIG. 18 shows the CDR regions of antibodies that bind to LTA (also identified in SEQ ID NOs: 1-8). These CDR sequences were identified based on the Kabat immunoglobulin classification nomenclature. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS [0066]
  • Antibodies which bind to one or more components of infectious bacteria (such as S. aureus), the methods of making said antibodies, and the methods of using said antibodies are provided. In several embodiments, the antibodies are used either as single agents or combined in a cocktail.
  • Embodiments of the antibodies exhibit at least one of LTA binding, bacterial binding, in vivo killing of bacteria, and in vitro killing of bacteria.
  • the bacteria are any bacteria recognized and/or killed by the antibodies, for example, Staphylococcus, such as S. aureus, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, and/or S. warneri.
  • the bacteria comprise drug-resistant, for example oxicillin resistant strains.
  • a method of passive immunization is used to prophylactically protect a mammal against bacterial infection.
  • Passive immunization shall be given its ordinary meaning and shall also mean the introduction of antibodies, for example, from an individual with active immunity, or of genetically- engineered or synthetic antibodies, to treat infection.
  • Passive immunization shall also include the administration of one or more antibodies, or fragments thereof, to confer immunity to a specific pathogen or toxin.
  • passive immunization is used when there is a high risk of infection and/or when there is insufficient time for a patient to develop an immune response.
  • passive immunization is used to reduce the symptoms of an infection or an immunosuppressive disease.
  • Fully human antibodies shall mean antibodies with 100% human nucleic acid or protein sequences.
  • a fully human monoclonal antibody to bacteria may be generated by administering human cells (typically from one or more human donors exposed to bacteria) to an immunocompromised animal, isolating a lymphocytic cell from that animal, and fusing the lymphocytic cell with a fusion partner, which then produces a fully human monoclonal antibody which recognizes at least a portion of the bacteria (such as LTA).
  • the terms “antibody” and “immunoglobulin” shall be used interchangeably. Antibodies for immunizing mammals and/or for treating mammals are provided in some embodiments of the invention. In some embodiments, the mammals include humans, but non-human mammals, livestock, and domesticated mammals may also benefit from certain embodiments of the invention.
  • blood cells from donors who have been exposed to bacteria are obtained. Such exposure may have occurred naturally through exposure, or may have occurred by vaccination. Moreover, in one embodiment, exposure may have occurred decades, years, or days prior to obtaining the donor's blood cells.
  • the "memory" of said exposure is captured or recalled and is selectably expanded by immunizing the engrafted SCID mice.
  • said recall technology is used to generate human monoclonal antibodies.
  • the use of human blood cells that have been "pre-exposed" to bacteria (such as S. aureus), or another target antigen yields several advantages. These advantages include the generation of antibodies with higher affinity, higher specificity, and more potent neutralization capabilities. Methods of making fully human antibodies according to U.S. Patent Nos. 5,476,996 and 5,698,767 are herein incorporated by reference in their entirety.
  • peripheral blood mononuclear cells are obtained from a donor.
  • other cell types are obtained, including but not limited to lymphocytes, splenocytes, bone marrow, lymph node cells.
  • the blood cells are administered to an immunocompromised or immuno-deficient animal.
  • the animal is a SCID mouse.
  • the animal is irradiated.
  • the animal's immune response is characterized using a test bleed.
  • the generated antibodies are screened and isolated.
  • the lymphocytic cells are transformed with EBV.
  • one or more booster injections of antigen are administered to the immuno-compromised animal.
  • one or more injections of anti-human CD8 is administered to the animal.
  • a double selection method to select against undesirable cells is used, including, but not limited to using HAT and ouabain.
  • the hybridoma fusion partner is a mouse myeloma or a mouse-human heteromyeloma.
  • the invention comprises one or more of the following human antibodies or fragments thereof: AVP-15C8S (FIG. 9, SEQ ID NO: 1 and SEQ ID NO: 2), AVP-6B10 (FIG. 10, SEQ ID NO: 3 and SEQ ID NO: 4), AVP-3E7 (FIG. 11, SEQ ID NO: 5 and SEQ ID NO: 6), and AVP-V-7-27 (FIG. 12, SEQ ID NO: 7 and SEQ ID NO: 8).
  • the sequences are not fully human and comprise a purified or isolated antibody having or encoded by one or more of the following amino acid or nucleotide sequences: SEQ ID NOs: 1-64. Formulations comprising these antibodies, or fragments thereof, are also provided.
  • the invention comprises a method of making any of the antibodies or any combination of the antibodies described herein. Additionally, any of the antibodies described herein may be provided in an isolated or purified form.
  • the invention comprises a method of using any of the antibodies or any combination of the antibodies described herein in the prevention, diagnosis, or treatment of wide spectrum of bacterial infections.
  • a monoclonal antibody of several embodiments can be administered as a pharmaceutical composition or formulation.
  • the antibody can be administered by several different routes, including but not limited to: parenterally, topically, and orally.
  • parenterally shall be given its ordinary meaning and shall also include subcutaneous, intravenous, intraarterial, injection, and/or infusion techniques, without limitation.
  • the antibody is administered intramuscularly.
  • topically shall be given its ordinary meaning and shall also encompasses administration rectally and by inhalation spray, as well as the more common routes of the skin and the mucous membranes of the mouth and nose.
  • one or more antibodies are administered via a syringe, patch, inhalants, and/or oral formulation.
  • Pre-prepared and pre-dosed antibody formulations can be available in kits so that individuals have easy and quick access to the antibody.
  • Such pre-dosed formulations e.g., syringes, patches, sprays, oral compositions
  • the appropriate dosage to be administered may be varied so as to administer an amount of a the antibody that is effective to achieve the desired therapeutic response for a particular patient.
  • the selected dosage level will depend upon the activity of the particular agent the route of administration, the severity of the condition being treated, and the condition and prior medical history of the patient being treated.
  • the effective daily dose may be divided into multiple doses for purposes of administration, e.g., about two to four separate doses per day. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, diet, time and route of administration, combination with other drugs and the severity of the particular disease being treated.
  • a therapeutically effective amount of the antibody has a plasma concentration of from about 0.01 ⁇ g/mL to about 500 ⁇ g/mL, from about 1 ⁇ g/mL to about 100 ⁇ g/mL.
  • therapeutically effective includes effectiveness in treatment of infections and/or prophylaxis.
  • Some embodiments of the dosage are from about 0.1 mg/kg to about 50 mg/kg, from about 0.2 mg/kg to about 30 mg/kg, or from about 0.5 mg/kg to about 20 mg/kg.
  • the antibody is administered in one or more doses over one or several days, according to some embodiments. Dosages are adjusted as known in the art in embodiments in which the active agent is a fragment of a monoclonal antibody or a conjugate.
  • the pharmaceutical formulation can be in a variety of forms, including, but not limited to, injectable fluids, suppositories, powder, tablets, capsules, syrups, suspensions, liquids, and elixirs.
  • the route is by injection.
  • an antibody preparation is pre-packaged in a self-injectable device, such as a syringe.
  • Embodiments of therapeutic formulations of the antibodies are prepared for storage and/or administration by mixing an antibody of a desired degree of purity with one or more optional pharmaceutically acceptable carriers, excipients, and/or stabilizers (see, for example, Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso R. Gennaro (ed),. (2000)).
  • the formulations may be stored as lyophilized formulations or aqueous solutions.
  • Acceptable carriers, excipients, and/or stabilizers are nontoxic at the dosages and concentrations employed. In some embodiments, the carriers, excipients, and/or stabilizers do not cause and/or reduce undesirable physiological effects, for example, nausea, dizziness, gastric upset, and the like.
  • Suitable carriers, excipients, and/or stabilizers are known in the art, and may include buffers ⁇ e.g., phosphate, citrate, and/or other organic acids); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, and/or lysine); monosaccharides, disaccharides, and other carbohydrates and sugar alcohols ⁇ e.g., glucose, mannose, sucrose, mannitol, trehalose, sorbitol, and/or dextrins); salts ⁇ e.g., sodium chloride, sodium salts, potassium salts, magnesium salts, calcium salts, acetates, lactates, chlorides); and/or surfactants ⁇ e.g., non-ionic surfactants, polysorbates (TWEEN®, ICI), ethylene oxide-propylene oxide copolymers (PLURONIC®, BASF), and/or polyethylene glycol (PEG)).
  • Embodiments of the formulation herein may also contain more than one active compound as desired for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • one or more antibodies or fragments described herein are provided in combination with one or more of the following antibiotics: benzylpenicillin, cloxacillin, amoxicillin, amoxicillin plus clavulanic acid, cephalonium, cefoperazone, erythromycin, tylmicosin, kanamycin, tetracycline, and vancomycin
  • One or more of the active compounds may also be microencapsulated, for example, by coacervation or by interfacial polymerization, for example, in hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g, liposomes, albumin microspheres, microemulsions, nano-particles, and/or nanocapsules) and/or in macroemulsions.
  • colloidal drug delivery systems e.g, liposomes, albumin microspheres, microemulsions, nano-particles, and/or nanocapsules
  • formulations to be used for in vivo administration are sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes.
  • Sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, in the form of shaped articles (e.g., films, microcapsules).
  • sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, and/or poly- ⁇ -(-)- 3-hydroxybutyric acid.
  • Embodiments of polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release the active ingredient over an extended period, for example, up to or over 100 days.
  • Embodiments of certain hydrogel compositions release the active ingredient over a shorter time period.
  • Sustained release formulations may be stabilized by any suitable method known in the art, for example, by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using additives, and/or using specific polymer matrix compositions.
  • Embodiments of the pharmaceutical formulations comprise pharmaceutically acceptable salts of one or more of the active agents.
  • Pharmaceutically acceptable salts include the acid addition salts formed between one or more free amino groups of the antibody and one or more acids, for example, inorganic acids (e.g., hydrochloric acid, phosphoric acid, and the like) and/or organic acids (e.g., acetic acid, tartaric acid, mandelic acid, and the like).
  • Salts may be formed between one or more free carboxyl groups of the and one or more bases, for example, inorganic bases (e.g., sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, ferric hydroxide, and the like) and/or organic bases (e.g., organic amines, isopropylamine, trimethylamine, 2- ethylaminoethanol, histidine, and the like).
  • inorganic bases e.g., sodium hydroxide, potassium hydroxide, ammonia, calcium hydroxide, ferric hydroxide, and the like
  • organic bases e.g., organic amines, isopropylamine, trimethylamine, 2- ethylaminoethanol, histidine, and the like.
  • kits for identifying the presence of bacteria such as S. aureus and/or other bacteria
  • a monoclonal antibody which specifically recognizes at least a portion of a component of bacteria is provided.
  • a sample is contacted with a monoclonal antibody which specifically recognizes at least a portion of a component of bacteria. If bacteria are present, then the binding of the bacteria with the monoclonal antibody can be determined. In this manner, certain embodiments of the invention are particularly useful in the diagnosis of bacterial infection.
  • kit as used herein shall be given its ordinary meaning and shall also include a compilation, collection, or group of materials used for a common goal or purpose.
  • a kit to test for the presence or absence of bacteria includes one or more of the following: an antibody, a swabbing material, gloves, an assay kit, and instructions (e.g., instructions for use or other types of instructions).
  • the invention comprises one or more amino acid and/or nucleotide sequences.
  • the following chart provides a list of certain sequences identified herein: Amino Acid SEQ ID NO Nucleotide SEQ ID NO Antibody or Fragment Corresponding FIG.
  • FIG. 9A 1 33 15C8S VH FIG. 9A 2 34 15C8S VK FIG. 9B 3 35 6B10 VH FIG. 1OA 4 36 6B10 VK FIG. 1OB 5 37 3E7 VH FIG. HA 6 38 3E7 VK FIG. HB 7 39 V-7-27 VH FIG. 12A
  • the invention comprises sequences with one or more variations or modifications as compared to amino acid sequences SEQ ID NOs: 1-8 and/or nucleotide sequences SEQ ID NOs: 33-40.
  • Variants include, but are not limited to, amino acid, codon, or base pair substitutions; additions; and deletions, as well as modifications (e.g., glycosylation, phosphorylation, methylation).
  • Variations or modifications of the nucleotide sequence may or may not result in modifications of the encoded amino acid sequence.
  • nucleotide changes do not substantially alter the properties or activities of the polynucleotide or polypeptide.
  • variants include alterations in the amino acid or peptide sequences.
  • Other variants include recombinant antibodies comprising at least two of the sequences of SEQ ID NOs: 1-8.
  • amino acid substitutions are based on the relative similarity of the amino acid side-chain substituents, such as hydrophilicity, hydrophobicity, size, charge, etc.
  • Substitutions include, but are not limited to, substitution between the following pairs of amino acids: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • the invention comprises nucleic acid or amino acid sequences that are less than 100% homologous to the sequences listed herein.
  • the invention comprises a DNA or amino acid sequence that is from about 80% to about 99% identical to at least one of the sequences identified as SEQ ID NOs: 1-8 and/or SEQ ID NOs: 33-40.
  • the invention comprises a DNA sequence that has partial homology (e.g., about 80%, 90%, or 95%) with the DNA sequences shown as SEQ ID NOs: 33-40.
  • the invention comprises an amino acid sequence that has partial homology (e.g., about 80%, 90%, or 95%) with the amino acid sequences shown as SEQ ID NOs: 1-8.
  • the sequences having homology to the sequences listed herein bind to the LTA region to achieve a therapeutic result.
  • the invention includes an antibody comprising three complementarity determining regions (CDR) shown in any one of SEQ ID NOs: 1- 8, also shown in FIG. 18 as SEQ ID NOs: 9-32. Corresponding nucleotide sequences are provided as SEQ ID NOs: 41-64. Each of SEQ ID NOs 1-8 comprises three CDR regions and framework regions.
  • the invention comprises an antibody comprising CDRlH, CDR2H, and CDR3H or CDRlL, CDR2L, and CDR3L of Antibody 15C8S, 6B10, 3E7, or V-7-27.
  • the invention comprises an isolated or purified antibody comprising CDRlH, CDR2H, and CDR3H and CDRlL, CDR2L, and CDR3L of Antibody 15C8S, 6B10, 3E7, or V-7-27.
  • Some embodiments comprise an antibody comprising at least one of CDRlL, CDR2L, and CDR3L of Antibody 15C8S, 6B10, 3E7, or V-7-27, that is, alone or in combination. Some embodiments comprise a recombinant antibody comprising at least two of CDRlL, CDR2L, and CDR3L of Antibody 15C8S, 6B10, 3E7, or V-7-27.
  • Some embodiments comprise an antibody comprising at least one of CDRlH, CDR2H, and CDR3H of Antibody 15C8S, 6B10, 3E7, or V-7-27, that is, alone or in combination. Some embodiments comprise a recombinant antibody comprising at least two of CDRlH, CDR2H, and CDR3H of Antibody 15C8S, 6B10, 3E7, or V-7-27.
  • the invention comprises an antibody comprising any one of the CDR sequences shown in FIG. 18 as SEQ ID NOs: 9-32. In yet another embodiment, the invention comprises an antibody comprising at least two, three, or six of the CDR sequences shown in FIG. 18.
  • the invention include sequences that have at least partial homology to the CDR regions identified herein and which bind to LTA.
  • the invention comprises method of screening for variants.
  • the invention comprises substituting one or more hypervariable region (CDR) residues of any one of SEQ ID NOs: 1-8, also shown in FIG. 18 as SEQ ID NOs: 9-32, and selecting for improved characteristics relative to the parent antibody or sequence.
  • CDR hypervariable region
  • phage display techniques are used to produce variants.
  • the antibody comprise at least one constant region and at least one variable region.
  • the constant region of the antibody is believed to be responsible for effector functions through binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • the constant region is of any suitable isotype known in the art.
  • the light chain comprises a polypeptide sequence substantially identical to a polypeptide sequence encoded by a human C L nucleic acid sequence, or a portion thereof, or a nucleic acid sequence encoding the same.
  • the heavy chain comprises a polypeptide sequence substantially identical to a polypeptide sequence encoded by human C H sequence, or a portion thereof, or a nucleic acid sequence encoding the same.
  • Other embodiments comprise a variant constant region or portion thereof, for example, comprising one or more amino acid substitutions, insertions, and/or deletions.
  • Other embodiments do not comprise a constant region.
  • formulations comprising one or more of the antibodies disclosed herein are used for coating or washing medical equipment (including but not limited to catheters, tubing, and IV bags) and implantable medical devices. Topical formulations comprising one or more of the antibodies disclosed herein may also be used.
  • aureus (type 8) were detectable in serum of 20 unrelated blood donors (FIG. IA). The reactivity was not due to simple stickiness of antibodies since there was no signal in plates that were not coated with bacteria (data not shown). The same samples were also tested for activity in an opsonophagocytic killing assay to detect functional antibodies. Only a small fraction of the sera showed substantial killing activity in this assay (FIG. IB). Moreover, there was no direct correlation between killing activity and antibody level detected by ELISA since samples with highest killing activity had low titers measured by ELISA and several samples with high ELISA titers showed low killing activity.
  • FIG. 2A Cross-reactivity with LTA isolated from three different streptococcus strains (S. pyogenes, S. sanguis, and S. faecialis) and Bacillus subtilis was also tested (FIG. 2B).
  • AVP-6B10 showed strong reactivity with LTA isolated from S. aureus and B. subtilis.
  • AVP-6B10 also binds to LTA on S. epidermidis (Fig 2C).
  • AVP-6B10 and AVP-V-7-27 were also examined by immunofluorescence staining using an FITC-labeled F(ab') 2 fragment of goat anti-human IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) for detection (FIG. 3).
  • AVP-21D9 which recognizes an unrelated antigen, served as negative control.
  • Peripheral blood mononuclear cells were enriched from whole blood of healthy donors by density gradient centrifugation using Histopaque, 1077-1 (Sigma, St. Louis, MO).
  • Histopaque 1077-1
  • One of skill in the art will understand that other types of cells can also be used in accordance with several embodiments of the present invention.
  • one unit of blood from donors was obtained.
  • Female or male SCID/bg 10-12 week old mice were each engrafted with 2-3 x 10 7 isolated human PBMC mixed with 2 ⁇ g of antigen in 100 ⁇ L of PBS. The human cells and antigen mixture was directly injected into a mouse spleen.
  • Splenocytes were harvested on day 8 from those mice showing positive test bleeds in indirect ELISA.
  • Human hybridomas were generated from these splenocytes in separate fusions using a murine myeloma P3x63Ag8.653 with PEG- 1500 (Sigma, St. Louis, MO) as described (Kearney, J. F., A. Radbruch, et al. (1979). "A new mouse myeloma cell line that has lost immunoglobulin expression but permits the construction of antibody-secreting hybrid cell lines.” J. Immunol. 123(4): 1548-50.), with the modification that the P3x63Ag8.653:lymphocyte ratio for fusion was between 1:3-1 :5.
  • P3x63Ag8.653 was used in this exemplary method, one skilled in the art will understand that several fusion partners can be used in accordance with various embodiments of the current invention, including, but not limited to, cells derived from the mouse myeloma MOPC2, triomas, etc. Eleven to 14 days after fusion, hybridoma supernatants from 96 well plates were tested by indirect ELISA.
  • Human B cells were selected by magnetic beads (B-cell Negative Isolation Kit, Dynal Biotech Inc., Oslo, Norway) from human PBMC isolated by Histopaque density gradient centrifugation (1077-1 Sigma, St. Louis, MO). Cell surface IgM positive cells were removed by incubating B cells on the anti-human IgM antibody (Jackson ImmunoResearch, West Grove, PA) coated Petri dish for 15 min at room temperature. Unbound B cells were recovered and resuspended in IMDM medium supplemented with 30% of B95-8 conditioned medium as a source of Epstein Barr Virus (EBV), 10% bovine IgG depleted FBS, 10% Hybridoma Cloning Factor (IGEN, Gaithersburg, MD) and antibiotics. Cells were cultured at 10-50 cells per well on a NHLF feeder layer. Two to 3 weeks later, supernatants from 96 well plates were tested by indirect ELISA.
  • BBV Epstein Barr Virus
  • IGEN Hybridoma Cloning Factor
  • Variable region of immunoglobulin cDNAs were cloned from B cells derived from SCID mice engrafted with human PBMC and immunized with antigen(s) or transformed by EBV.
  • Total RNA was prepared from specific ELISA positive cells using RNeasy Mini Kit (Qiagen, Valencia, CA). Mixture of VH and VL cDNAs were synthesized and amplified in a same tube using One-Step RT-PCR Kit (Qiagen, Valencia, CA). Cycling parameters were 50 0 C for 35 min; 95 0 C for 15 min; 35 cycles of 94 0 C for 30 sec, 52 0 C for 20 sec and 72 0 C for 1 min 15 sec; and 72 0 C for 5 min.
  • Cycling parameters were 1 cycle of 94 0 C for 2 min, 60 °C for 30 sec, and 68 0 C for 45 sec; 35 cycles of 94 0 C for 40 sec, 54 0 C for 25 sec, and 68 0 C for 45 sec; and 68 0 C for 5 min.
  • Each specific PCR product was separately purified, digested with restriction enzymes, and subcloned into appropriate mammalian full-length Ig expression vectors as described below. Heavy or light variable region cDNA was subcloned into Xenerex Ig expression cassette separately and transiently co-expressed in CHO cells.
  • primers for nested PCR contain appropriate restriction enzyme sites to facility cloning into expression vectors.
  • VH ⁇ PCR products are digested with BsrG I and Apa I and ligated into pEEGl .l vector that is linearized by SpI I and Apa I double digestion.
  • VLK PCR products are digested with Age I and Xho I and ligated into pEEKl.l vector linearized by Xma I and Xho I double digestion.
  • VL ⁇ PCR products are digested with Apa I and Xho I and ligated into pEELg vector linearized by Apa I and Xho I double digestion.
  • CHO-Kl cells were transfected with different combinations of IgG and IgK or IgL cDNAs using Lipofectamine-2000 (Invitrogen, Carlsbad, CA).
  • the antigen specific cDNA clones were identified by screening supernatants after transient co-transfection of heavy and light chains and detecting secreted antibodies by indirect ELISA on LTA coated plates. Multiple positive cDNA clones were sequenced with the ABI 3700 automatic sequencer (Applied Biosystems, Foster City, CA) and analyzed with Sequencher v4.1.4 software (Gene Codes, Ann Arbor, MI). For antibodies that passed the selection criteria, a dual expression vector was constructed by ligating the heavy and light chain into a single expression cassette, and a stable CHO cell line expressing individual anti-Staphylococcus antibody was established.
  • Ig heavy chain or light chain expression vector were double digested with Not I and Sal I, and then both fragments were ligated to form a double gene expression vector.
  • CHO-Kl cells in 6 well-plates were transfected with the double gene expression vector using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 24 hrs transfection cells were transferred to a 10 cm dish with selection medium (D.MEM supplemented with 10% dialyzed FBS, 60 ⁇ M L-methionine Sulphoximine (MSX), penicillin/streptomycin, GS supplement). Two weeks later MSX resistant transfectants were isolated and expanded. Anti-LTA antibody high producing clones were selected by measuring the supernatant with LTA specific ELISA assay. MSX concentration was increased from 50 ⁇ M to 100 ⁇ M to enhance the antibody productivity.
  • a stable cell line was cultured in 10% dialyzed FBS in ExCeIl 302 serum-free medium (JRH, 1000M) with Ix GS (JRH, 100 M), penicillin/streptomycin, and 60-100 ⁇ M L-Methionine Sulphoximine (Sigma). Cells were treated with trypsin (Omega) and split 1 :5. The culture medium was switched to 5% FBS containing media and the cells were cultured 2 days. After the cells were adapted to growing in 5% FBS containing media, the media was changed to the medium containing 2.5% dialyzed FBS for 1-2 days, then to 100% serum free media.
  • the protein content in eluted fractions was determined by absorbance at 280 nm, the fractions containing antibody were pooled and dialyzed against 20 mM Tris, 150 mM NaCl, pH 7.4 (2 x 500 volumes), and filter sterilized through a 0.2 ⁇ m filter. An aliquot of sterile 10% Tween-80 was added to a yield a final concentration of 0.01%. The antibody was further characterized by SDS- PAGE and the purity exceeded 95%.
  • IgG l ⁇ myeloma protein (Athens Research, Athens, GA) served as an internal calibrator for comparison.
  • Diluted test samples 50 ⁇ L were transferred to the wells of the assay plate and incubated for one hour at room temperature. Plates were washed as before and 50 ⁇ L of the detecting antibody (1 :4000 in PBS with 1 mg/mL BSA).
  • Goat anti-human kappa-HRP Southernn Biotechnology Associates, Inc., Birmingham, AL was added and incubated for one hour at room temperature.
  • Opsonophagocytic killing mediated by anti-pathogen antibodies, complement, and phagocytes e.g., neutrophils, monocytes, and macrophages
  • phagocytes e.g., neutrophils, monocytes, and macrophages
  • In vitro opsonophagocytosis is believed to be a reliable surrogate for subsequent clinical efficacy in vivo of the specific antibodies.
  • binding of the antibodies to bacteria triggers uptake by phagocytes, where the bacteria are killed.
  • aureus (Type 8, Type 5 or MRSA BAA- 41 strain) was incubated with or without various dilutions of recombinant anti- staphylococcus antibody at room temperature for 30 min in a 96 well plate.
  • human complement final concentration 1%) was added to each well and freshly isolated PMNs were added to the mixture immediately afterwards and incubated at 37 °C for 60-90 min with shaking.
  • the content of each well was plated on agar plates that were incubated overnight at 37 0 C to allow the remaining viable bacteria to form visible colonies that can be counted.
  • the samples containing bacteria, PMN and human complement alone (without neutralizing antibodies) showed typically approximately 200 colonies, which was used to define 0% killing.
  • AVP-6B10, 3E7, and V-7-27 showed > 50% killing activity against S. aureus type 8 at concentrations > 2 ⁇ g/mL, while AVP-15C8S required > 50 ⁇ g/mL in this assay (FIG. 4).
  • Potent killing activity of several antibodies was also observed when S. aureus type 5 or a MRSA strain (BAA-41) was used in this assay, while AVP-21D9, an antibody that recognizes an unrelated antigen (protective antigen of Bacillus anthracis) failed to show bacterial killing activity (FIG. 5).
  • IgGi human immunoglobulin G
  • IgG 2 IgG 3
  • IgG 4 IgG 4
  • IgGi human immunoglobulin G
  • the four IgG isotypes differ from each other with respect to their effector functions. It is well known that antibodies of the IgG 3 isotype have better complement activation and higher affinity to Fc ⁇ receptor (Fc ⁇ R), while IgG 2 antibodies only exhibit weak effector functions and IgG 4 antibodies essentially lack those activities. Therefore, IgG 3 and IgG 2 antibodies were generated without changing the variable region by switching the heavy chain constant region from IgGi to the corresponding IgG 3 or IgG 2 coding region. In brief, cDNA encoding IgG 3 and IgG 2 heavy chain constant regions were PCR-cloned from EBV-transformed B cells and used to replace the IgGi sequence in the Xenerex expression vector.
  • a mouse S. aureus mediated acute lethal infection model was established to evaluate lead candidates in vivo. Based on the in vitro OPKA results (Table 3) specific antibody producing CHO cell lines were adapted to serum free medium and cultured in Integra flasks or a WaveTM Biotechnology bioreactor. Antibodies were purified from supernatant using either a Protein A (for IgGi) or Protein G (for IgG 3 ) column. AVP-3E7/G3 showed partial protection in this acute lethal model against the MRSA strain BAA-41 (FIG. 6). All mice treated with AVP-21D9 control antibody died within 24 hours post infection, while 40% of the AVP-3E7/G3 treated mice survived the challenge.
  • mice infected with S. aureus type 5 bacteria and treated with AVP-V-7-27 Similar results were obtained in mice infected with S. aureus type 5 bacteria and treated with AVP-V-7-27 (FIG. 7).
  • In vivo inactivation or killing of bacteria may be supplemented with one or more antibiotics, which may show additive or synergistic effects.
  • two or more antibodies may be used, showing either an additive or synergistic effect.

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Abstract

La présente invention concerne, dans certains modes de réalisation, des anticorps totalement humains qui se lient à Staphylococcus aureus et véhiculent une tuerie υpsυnυphagocytique en présence de cellules polymorphes nucléaires humaines et d'un complément humain. Les anticorps sont utiles pour le traitement ou la prophylaxie de diverses infections provoquées par S. aureus, y compris MRSA (S. aureus résistant à la méthicilline).
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US20040052779A1 (en) * 2001-12-21 2004-03-18 Stinson Jeffrey R. Opsonic monoclonal and chimeric antibodies specific for lipoteichoic acid of Gram positive bacteria

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