Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
  • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
  • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus

Definitions

• a biofilm is an aggregate of microorganisms in which the individual cells adhere to each other on a surface, usually embedded within a self-produced polymeric matrix.
• Biofilms have been found to be involved in a wide variety of microbial infections in the human body, including common conditions such as urinary tract infections, catheter infections, middle-ear infections, formation of dental plaque, gingivitis, as well as less common but more lethal conditions such as endocarditis, lung infections in cystic fibrosis, and infections of permanent indwelling devices, such as joint prostheses, heart valves and intrauterine devices. It has also been noted that bacterial biofilms may impair cutaneous wound healing and reduce topical antibacterial efficiency in healing or treating infected skin wounds.
• PNAG poly-N-acetylglucosamine
• Staphylococcus epidermidis and S. aureus are the most frequent causes of nosocomial infections (e.g., on indwelling medical devices), which characteristically involve biofilms.
• Biofilm formation by S. epidermidis and S. aureus is particularly problematic in that it protects the bacteria from attack by antibiotics and the host immune system, thereby rendering the infection difficult to treat.
• the present invention provides methods for the treatment or prevention of microbial infections (e.g., nocosomial infection) in which the underlying pathology involves a PNAG- containing microbial biofilm.
• the methods of the invention generally involve administering to the subject an effective amount of an antibody that specifically binds to PNAG and disrupts or inhibits formation of PNAG-containing microbial biofilms.
• Such methods are particularly useful for the treatment of nosocomial staphylococcus (e.g., S.epidermidis and S. aureus) infections.
• the invention provides a method of preventing a nocosomial infection comprising, the method generally comprising: identifying a subject
• PNAG poly-N-acetyl glucosamine
• the nocosomial infection is a lung infection, joint infection, endocardial infection, skin infection, soft tissue infection, or septicemia.
• the antibody is administered between about 0 and 240 hours prior to the medical procedure.
• the medical procedure is the installation of a surgical implant in the subject, e.g., a stent, catheter, cannula, prosthesis, or pace-maker.
• a surgical implant e.g., a stent, catheter, cannula, prosthesis, or pace-maker.
• the antibody is administered systemically. In other embodiments, the antibody is administered locally to the site of implantation of the surgical implant.
• the antibody is coated on the surgical implant or adhered to the surgical implant.
• the antibody is administered in a controlled release formulation.
• the biofilm comprises Staphylococcus, e.g., S. epidermidis and S. aureus.
• the antibody is a human antibody.
• the antibody comprises a VH domain comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1 , 2, and 3, respectively.
• the antibody comprises a VL domain comprising the LCDR1 , LCDR2, and LCDR3 amino acid sequences set forth in SEQ ID Nos: 4, 5, and 6, respectively.
• the antibody comprises the VH domain sequence set forth in SEQ ID No: 7.
• the invention provides a method of inhibiting formation of a poly N-acetyl glucosamine (PNAG) -containing microbial biofilm on a surgical implant in a subject (e.g., a human subject), the method generally comprising administering to the subject an effective amount of an antibody that specifically binds to PNAG prior to implantation of the surgical implant into the subject.
• PNAG poly N-acetyl glucosamine
• the surgical implant is a stent, catheter, cannula, prosthesis, or pace-maker.
• the antibody is administered between about 0 and 240 hours prior to the medical procedure.
• the antibody is administered systemically. In other embodiments, the antibody is administered locally to the site of implantation of the surgical implant.
• the antibody is coated on the surgical implant or adhered to the surgical implant.
• the antibody is administered in a controlled release formulation.
• the biofilm comprises Staphylococcus, e.g., S. epidermidis and S. aureus.
• the antibody is a human antibody.
• the antibody comprises a VH domain comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1 , 2, and 3, respectively.
• the antibody comprises a VL domain comprising the
• the antibody comprises the VH domain sequence set forth in SEQ ID No: 7.
• the invention provides a method of inhibiting formation of PNAG- containing microbial biofilm on a substrate comprising contacting the substrate with an effective amount of an antibody that specifically binds to PNAG.
• the biofilm comprises Staphylococcus, e.g., S. epidermidis and S. aureus.
• the antibody is a human antibody.
• the antibody comprises a VH domain comprising the HCDRl, HCDR2, and HCDR3 amino acid sequences set forth in SEQ ID Nos: 1 , 2, and 3, respectively.
• the antibody comprises a VL domain comprising the LCDR1 , LCDR2, and LCDR3 amino acid sequences set forth in SEQ ID Nos: 4, 5, and 6, respectively.
• the antibody comprises the VH domain sequence set forth in SEQ ID No: 7.
• Figure 1 depicts the results of in vitro assays measuring biofilm formation of S.
• Figure 2 depicts the results of in vitro assays measuring biofilm formation of S.
• Figure 3 depicts the results of in vitro assays measuring biofilm formation of S.
• the present invention provides methods for the treatment or prevention of microbial infections (e.g., nocosomial infection) in which the underlying pathology involves a PNAG- containing microbial biofilm.
• the methods the invention generally involve administering to the subject an effective amount of an antibody that specifically binds to PNAG and disrupt or inhibit formation of PNAG-containing microbial biofilms.
• Such methods are particularly useful for the treatment of nosocomial staphylococcus (e.g., S.epidermidis and S. aureus) infections.
• poly-N-acetyl glucosamine or "PNAG” refer to a polymer of N-acetyl glucosamine monomers linked via a beta 1-6 linkage. The terms also encompass partially or fully deacylated poly-N-acetyl glucosamine.
• PNAG-containing microbial bio film refers to sessile aggregate of microorganisms embedded in a PNAG matrix.
• nocosomial infection refers to infection acquired in a hospital or from a medical procedure performed inside or outside of a hospital.
• exemplary nocosomial infections include sepsis (bloodstream infection), surgical site infection, or hospital-acquired pneuomia.
• preventing a nocosomial infection refers to an inhibition or reduction in the severity of an infection.
• the term "antibody” refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM).
• Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region.
• the heavy chain constant region comprises three domains, CHI , CH2 and CH3.
• Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region.
• the light chain constant region comprises one domain (CLI).
• VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
• CDRs complementarity determining regions
• FR framework regions
• Each VH and VL is composed of three CDRs and four FRs, arranged from amino -terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
• antigen-binding portion of an antibody include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.
• Antigen- binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains.
• Non-limiting examples of antigen- binding portions include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)).
• CDR complementarity determining region
• Other engineered molecules such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression "antigen-binding portion.”
• CDR or "complementarity determining region” means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al, J. Mol. Biol. 196:901 -917 (1987) and by MacCallum et al., J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison.
• the term "CDR” is a CDR as defined by Kabat, based on sequence comparisons.
• framework (FR) amino acid residues refers to those amino acids in the framework region of an Ig chain.
• framework region or "FR region” as used herein, includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Kabat definition of CDRs). Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs.
• the term “specifically binds to” refers to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with an Kd of at least about 1 x 10 ⁇ 6 M, 1 x 10 "7 M, 1 x 10 "8 M, 1 x 10 "9 M, 1 x 10 "10 M, 1 x 10 "11 M, 1 x 10 "12 M, or more, and/or bind to an antigen with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
• the term "antigen” refers to the binding site or epitope recognized by an antibody or antigen binding portion thereof.
• the term “effective amount” refers to that amount of an antibody or an antigen binding portion thereof that binds PNAG, which is sufficient to effect treatment, prognosis or diagnosis of a PNAG-containing microbial biofilm, as described herein, when administered to a subject.
• a therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• the dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about 20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng to about 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about 6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000 mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg, about 1 ug to about 3,500 mg, about 5 ug to about 3,000 mg, about 10 ug to about 2,600 mg, about 20 ug to about 2,575 mg, about 30 ug to about 2,550 mg, about 40 ug to about 2,500 mg, about 50 ug to about 2,475 mg, about 100 ug to about 2,450 mg, about 200 ug to about 2,425 mg, about 300 ug to about 2,000 mg, about 400 ug to about 1,175
• the term "subject” includes any human or non-human animal.
• PNAG-containing biofilm can be produced by a variety of microbes, including bacteria and fungi. In general, any PNAG- containing microbial biofilm can be disrupted or inhibited using the methods of the invention.
• PNAG-containing bio films are commonly formed by, for example, Staphylococci (e.g., S. epidermis, S. aureus (e.g., Multi Drug Resistant S. aureus), S. carnosus, and S. haemolyticus).
• Staphylococci e.g., S. epidermis, S. aureus (e.g., Multi Drug Resistant S. aureus), S. carnosus, and S. haemolyticus.
• PNAG-containing bio films include, without limitation, S. epidermis RP62A (ATCC number 35984), S. epidermis RP12 (ATCC number 35983), S. epidermis Ml 87, S. carnosus TM300 (pCN27), S. aureus RN4220 (pCN27), and S. aureus MN8 mucoid.
• PNAG-containing bio films are also formed by other bacteria including but not limited to Pseudomonas aeruginosa, E. coli (e.g., E. coli 0157:H7 and E. coli CFT073), Yersinia pestis, Yersinia entercolitica, Xanthomonas axonopodis, Pseudomonas fluorescens,
• the invention provides methods for treating or preventing nosocomial infections by identifying a subject at risk of developing a poly-N-acetyl glucosamine (PNAG)-containing microbial biofilm from a medical procedure and administering to the subject an effective amount of an antibody that specifically binds to PNAG and inhibits PNAG-containing microbial biofilm formation.
• PNAG poly-N-acetyl glucosamine
• any medical procedure, whether performed inside or outside of a hospital, that confers a risk to a patient of developing a PNAG-containing microbial biofilm can be treated or prevented using the methods of the invention.
• medical procedures include, without limitation, surgery and implantation of a surgical device (e.g., catheter, cannula, prosthesis, respirator, replacement heart valve, and pace-maker).
• a surgical device e.g., catheter, cannula, prosthesis, respirator, replacement heart valve, and pace-maker.
• Exemplary surgical devices include, without limitation, central venous catheters; peritoneal dialysis catheters; orthopedic prostheses; orthopedic mesh; intracardiac devices such as artificial valves, pacemakers, and stents; cochlear implants; breast implants; endotracheal tubes; voice prostheses; intraocular lens
• Any antibody that binds to PNAG and inhibits formation of a PNAG-containing bacterial biofilm can be used in the methods of the invention.
• Exemplary antibody VH, VL and CDR amino acid sequences suitable for use in the invention are set forth in Table 1.
• the antibody, or antigen binding fragment thereof comprises one or more CDR region amino acid sequence selected from the group consisting of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 9, 10, 11, 12, 13, 14, 17, 18, 19, 20, 21, and 22.
• HCDR3, HCDR2 and HCDRl region amino acid sequences selected from the group consisting of:
• the antibody, or antigen binding fragment thereof comprises the LCDR3, LCDR2 and LCDRl region amino acid sequences selected from the group consisting of:
• the antibody, or antigen binding fragment thereof comprises the HCDR3, HCDR2, HCDRl , LCDR3, LCDR2 and LCDRl region amino acid sequences selected from the group consisting of:
• the antibody, or antigen binding fragment thereof comprises the VH region amino acid sequences set forth in SEQ ID NO: 7, 15 and/or 23. In other embodiments, the antibody, or antigen binding fragment thereof, comprises the VL region amino acid sequences set forth in SEQ ID NO: 8, 16, and/or 24.
• the antibody, or antigen binding fragment thereof comprises the VH and VL region amino acid sequences selected from the group consisting of: SEQ ID NO: 7 and 8; SEQ ID NO: 15 and 16; and SEQ ID NO: 23 and 24, respectively.
• Anti-PNAG antibodies may comprise one or more modifications. Such modified forms of anti-PNAG antibodies can be made using any techniques known in the art. i) Reducing Immunogenicity
• de-immunization can be used to decrease the immunogenicity of and antibody, or antigen binding portion thereof.
• the term "de- immunization” includes alteration of an antibody, or antigen binding portion thereof, to modify T cell epitopes (see, e.g., W09852976A1, WO0034317A2).
• VH and VL sequences from the starting antibody may be analyzed and a human T cell epitope "map" may be generated from each V region showing the location of epitopes in relation to complementarity-determining regions (CDRs) and other key residues within the sequence.
• CDRs complementarity-determining regions
• T cell epitopes from the T cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering activity of the final antibody.
• a range of alternative VH and VL sequences are designed comprising combinations of amino acid substitutions and these sequences are subsequently incorporated into a range of anti- PNAG antibodies or fragments thereof for use in the methods disclosed herein, which are then tested for function.
• Complete heavy and light chain genes comprising modified V and human C regions are then cloned into expression vectors and the subsequent plasmids introduced into cell lines for the production of whole antibody.
• the antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.
• Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfment and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production.
• the anti-PNAG antibodies, or fragments thereof bind to an Fc.gamma. receptor.
• anti- PNAG antibodies may comprise a constant region which is devoid of one or more effector functions (e.g., ADCC activity) and/or is unable to bind Fey receptor.
• Certain embodiments of the invention include anti-PNAG antibodies in which at least one amino acid in one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as reduced or enhanced effector functions, the ability to non-covalently dimerize, increased ability to localize at the site of a tumor, reduced serum half-life, or increased serum half-life when compared with a whole, unaltered antibody of approximately the same immunogenicity.
• certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein are domain deleted antibodies which comprise a polypeptide chain similar to an immunoglobulin heavy chain, but which lack at least a portion of one or more heavy chain domains.
• one entire domain of the constant region of the modified antibody will be deleted, for example, all or part of the CH2 domain will be deleted.
• an Fc domain employed in an anti-PNAG antibody is an Fc variant.
• the term "Fc variant” refers to an Fc domain having at least one amino acid substitution relative to the wild-type Fc domain from which said Fc domain is derived.
• the Fc variant of said human IgGl Fc domain comprises at least one amino acid substitution relative to said Fc domain.
• the antibodies may employ any art-recognized Fc variant which is known to impart an improvement (e.g., reduction or enhancement) in effector function and/or FcR binding.
• Said Fc variants may include, for example, any one of the amino acid substitutions disclosed in International PCT Publications WO88/07089A1, W096/14339A1 , WO98/05787A1 , W098/23289A1, W099/51642A1 , W099/58572A1 , WO00/09560A2, WO00/32767A1, WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2, WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,
• an anti-PNAG antibody of may comprise an Fc variant comprising an amino acid substitution which alters the antigen- independent effector functions of the antibody, in particular the circulating half-life of the antibody.
• Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder.
• Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time may be advantageous, e.g. for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods.
• Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women.
• other applications in which reduced FcRn binding affinity may be desired include those applications in which localization the brain, kidney, and/or liver is desired.
• an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of an Fc domain.
• the FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering).
• Exemplary amino acid substitutions which altered FcRn binding activity are disclosed in International PCT Publication No. WO05/047327 which is incorporated by reference herein.
• the antibodies, or fragments thereof comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).
• antibodies for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgGl or IgG4 heavy chain constant region, which is altered to reduce or eliminate glycosylation.
• an antibody may also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody.
• said Fc variant may have reduced glycosylation (e.g., N- or O- linked glycosylation).
• the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering).
• the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS.
• the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering).
• the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a T299A mutation (EU numbering).
• antibodies, or fragments thereof are modified to eliminate glycosylation.
• Such antibodies, or fragments thereof may be referred to as "agly” antibodies, or fragments thereof, (e.g. "agly” antibodies). While not being bound by theory, it is believed that "agly" antibodies, or fragments thereof, may have an improved safety and stability profile in vivo.
• Exemplary agly antibodies, or fragments thereof comprise an aglycosylated Fc region of an IgG4 antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital organs.
• antibodies, or fragments thereof comprise an altered glycan.
• the antibody may have a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated.
• the antibody may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.
• Covalent Attachment Anti-PNAG antibodies may be modified, e.g., by the covalent attachment of a molecule to the antibody such that covalent attachment does not prevent the antibody from specifically binding to its cognate epitope.
• the antibodies, or fragments thereof may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• Antibodies, or fragments thereof, may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalent and non-covalent conjugations) to polypeptides or other compositions.
• anti-PNAG antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387.
• Anti-PNAG antibodies may be fused to heterologous polypeptides to increase the in vivo half life or for use in immunoassays using methods known in the art.
• PEG can be conjugated to the anti-PNAG antibodies to increase their half- life in vivo. Leong, S. R., et al, Cytokine 16:106 (2001); Adv. in Drug Deliv. Rev. 54:531 (2002); or Weir et al, Biochem. Soc. Transactions 30:512 (2002).
• anti-PNAG antibodies can be fused to marker sequences, such as a peptide to facilitate their purification or detection.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)) and the "flag" tag.
• Anti-PNAG antibodies may be used in non-conjugated form or may be conjugated to at least one of a variety of molecules, e.g., to improve the therapeutic properties of the molecule, to facilitate target detection, or for imaging or therapy of the patient.
• Anti-PNAG antibodies can be labeled or conjugated either before or after purification, when purification is performed.
• anti-PNAG antibodies may be conjugated to therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEG.
• the present invention further encompasses anti-PNAG antibodies conjugated to a diagnostic or therapeutic agent.
• the anti-PNAG antibodies can be used diagnostically to, for example, monitor the development or progression of a immune cell disorder (e.g., CLL) as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen.
• Detection can be facilitated by coupling the anti-PNAG antibodies to a detectable substance.
• detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• bioluminescent materials include luciferase, luciferin, and aequorin;
• suitable radioactive material include 1251, 1311, l l lIn or 99Tc.
• Anti-PNAG antibodies for use in the diagnostic and treatment methods disclosed herein may be conjugated to cytotoxins (such as radioisotopes, cytotoxic drugs, or toxins) therapeutic agents, cytostatic agents, biological toxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, immunologically active ligands (e.g., lymphokines or other antibodies wherein the resulting molecule binds to both the neoplastic cell and an effector cell such as a T cell), or PEG.
• cytotoxins such as radioisotopes, cytotoxic drugs, or toxins
• an anti-PNAG antibody for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that decreases tumor cell growth.
• the disclosed compositions may comprise antibodies, or fragments thereof, coupled to drugs or prodrugs.
• Still other embodiments of the present invention comprise the use of antibodies, or fragments thereof, conjugated to specific biotoxins or their cytotoxic fragments such as ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin.
• the selection of which conjugated or unconjugated antibodyto use will depend on the type and stage of cancer, use of adjunct treatment (e.g., chemotherapy or external radiation) and patient condition. It will be appreciated that one skilled in the art could readily make such a selection in view of the teachings herein.
• radioisotopes include: 90Y, 1251, 1311, 1231, l l lln, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re.
• the radionuclides act by producing ionizing radiation which causes multiple strand breaks in nuclear DNA, leading to cell death.
• the isotopes used to produce therapeutic conjugates typically produce high energy alpha- or beta-particles which have a short path length. Such radionuclides kill cells to which they are in close proximity, for example neoplastic cells to which the conjugate has attached or has entered. They have little or no effect on non-localized cells. Radionuclides are essentially non- immunogenic.
• the route of administration of the antibodies, or fragments thereof, may be oral, parenteral, by inhalation or topical.
• parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration.
• the intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration are generally preferred. While all these forms of administration are clearly contemplated as being within the scope, a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip.
• a suitable pharmaceutical composition for injection may comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc.
• a buffer e.g. acetate, phosphate or citrate buffer
• a surfactant e.g. polysorbate
• a stabilizer agent e.g. human albumin
• the polypeptides can be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
• Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• pharmaceutically acceptable carriers include, but are not limited to, 0.01 -0.1M and preferably 0.05M phosphate buffer or 0.8% saline.
• Other common parenteral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present such as for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
• compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
• the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and will preferably be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• a coating such as lecithin
• surfactants for example, sodium bicarbonate
• chlorobutanol phenol, ascorbic acid, thimerosal and the like.
• isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition.
• Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
• sterile injectable solutions can be prepared by incorporating an active compound (e.g., an antibody by itself or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization.
• an active compound e.g., an antibody by itself or in combination with other active agents
• dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and freeze-drying, which yields a powder of an active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the preparations for injections are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. Further, the preparations may be packaged and sold in the form of a kit such as those described in co-pending U.S. Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which is incorporated herein by reference. Such articles of manufacture will preferably have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or at risk of obtaining a PNAG-conatining microbial biofilm.
• Effective doses of the stabilized antibodies, or fragments thereof, of the present invention, for the treatment of the above described conditions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
• the patient is a human, but non- human mammals including transgenic mammals can also be treated.
• Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
• the dosage may range, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the host body weight.
• dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the above ranges are also intended to be within the scope of the invention.
• Subjects can be administered such doses daily, on alternative days, weekly or according to any other schedule determined by empirical analysis.
• An exemplary treatment entails administration in multiple dosages over a prolonged period, for example, of at least six months. Additional exemplary treatment regimes entail administration once per every two weeks or once a month or once every 3 to 6 months.
• Exemplary dosage schedules include 1- 10 mg/kg or 15 mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kg weekly.
• two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dosage of each antibody administered may fall within the ranges indicated.
• Anti-PNAG antibodies, or fragments thereof can be administered on multiple occasions. Intervals between single dosages can be, e.g., daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of polypeptide or target molecule in the patient. In some methods, dosage is adjusted to achieve a certain plasma antibody or toxin concentration, e.g., 1-1000 ug/ml or 25-300 ug/ml. Alternatively, antibodies, or fragments thereof, can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half- life of the antibody in the patient. In general, humanized antibodies show the longest half-life, followed by chimeric antibodies and nonhuman antibodies.
• the antibodies, or fragments thereof can be administered in unconjugated form. In another embodiment, the antibodies can be administered multiple times in conjugated form. In still another embodiment, the antibodies, or fragments thereof, can be administered in unconjugated form, then in conjugated form, or vise versa.
• compositions containing the present antibodies or a cocktail thereof are administered to a patient not already in the disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactic effective dose.”
• prophylactic effective dose the precise amounts again depend upon the patient's state of health and general immunity, but generally range from 0.1 to 25 mg per dose, especially 0.5 to 2.5 mg per dose.
• a relatively low dosage is administered at relatively infrequent intervals over a long period of time.
• a relatively high dosage e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules
• a relatively high dosage e.g., from about 1 to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg being more commonly used for radioimmunoconjugates and higher doses for cytotoxin-drug conjugated molecules
• the patent can be administered a prophylactic regime.
• a subject can be treated with a nucleic acid molecule encoding a an anti-PNAG antibody (e.g., in a vector).
• a nucleic acid molecule encoding a an anti-PNAG antibody (e.g., in a vector).
• Doses for nucleic acids encoding polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ug to 10 mg, or 30-300 ug DNA per patient.
• Doses for infectious viral vectors vary from 10-100, or more, virions per dose.
• Therapeutic agents can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic and/or therapeutic treatment. Intramuscular injection or intravenous infusion are preferred for administration of an antibody.
• therapeutic antibodies, or fragments thereof are injected directly into the cranium.
• antibodies, or fragments thereof are administered as a sustained release composition or device, such as a MedipadTM device.
• Anti-PNAG antibodies can optionally be administered in combination with other agents that are effective in treating the disorder or condition in need of treatment (e.g., prophylactic or therapeutic).
• Preferred additional agents are those which are art recognized and are standardly administered for a particular disorder.
• Effective single treatment dosages (i.e., therapeutically effective amounts) of 90 Y- labeled antibodies range from between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi.
• Effective single treatment non-marrow ablative dosages of 1311- labeled antibodies range from between about 5 and about 70 mCi, more preferably between about 5 and about 40 mCi.
• Effective single treatment ablative dosages (i.e., may require autologous bone marrow transplantation) of 13 II- labeled antibodies range from between about 30 and about 600 mCi, more preferably between about 50 and less than about 500 mCi.
• an effective single treatment non-marrow ablative dosages of iodine-131 labeled chimeric antibodies range from between about 5 and about 40 mCi, more preferably less than about 30 mCi. Imaging criteria for, e.g., the l l lln label, are typically less than about 5 mCi.
• radiolabels are known in the art and have been used for similar purposes. Still other radioisotopes are used for imaging.
• additional radioisotopes which are compatible with the scope of the instant invention include, but are not limited to, 1231, 1251, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 1321, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211A 213BL
• alpha, gamma and beta emitters are all compatible with in the instant invention.
• radionuclides are compatible with a selected course of treatment without undue experimentation.
• additional radionuclides which have already been used in clinical diagnosis include 1251, 1231, 99Tc, 43 K, 52Fe, 67Ga, 68Ga, as well as 11 lln.
• Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Peirersz et al. Immunol. Cell Biol. 65: 111 -125 (1987)). These radionuclides include 188Re and 186Re as well as 199Au and 67Cu to a lesser extent.
• U.S. Pat. No. 5,460,785 provides additional data regarding such radioisotopes and is incorporated herein by reference.
• the antibodies, or fragments thereof can be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders.
• the disclosed antibodies, or fragments thereof will be formulated so as to facilitate administration and promote stability of the active agent.
• compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
• a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like.
• an antibody, conjugated or unconjugated to a therapeutic agent shall be held to mean an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., to ameliorate symptoms of a disease or disorder or to detect a substance or a cell.
• the polypeptide will be preferably be capable of interacting with selected immunoreactive antigens on neoplastic or
• compositions of the present invention may be administered in single or multiple doses to provide for a pharmaceutically effective amount of the polypeptide.
• anti-PNAG antibodies may be administered to a human or other animal in accordance with the aforementioned methods of treatment in an amount sufficient to produce a therapeutic or prophylactic effect.
• the anti- PNAG antibodies can be administered to such human or other animal in a conventional dosage form prepared by combining the antibody with a conventional pharmaceutically acceptable carrier or diluent according to known techniques. It will be recognized by one of skill in the art that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that a cocktail comprising one or more species of polypeptides according to the present invention may prove to be particularly effective.
• the invention provides methods for treating or preventing PNAG-containing bacterial biofilm by administering to a subject in need of thereof a pharmaceutical composition comprising one or more anti-PNAG antibody, or antigen binding fragment thereof.
• a pharmaceutical composition comprising one or more anti- PNAG antibodies, or antigen binding fragments thereof, are administered to a subject in combination with one or more additional therapeutic agents.
• the one or more additional therapeutic agents is administered concurrently with the
• Suitable additional therapeutic agents include antibacterial agents (e.g., antibiotics).
• antibacterial agents include, without limitation, penicillin G, penicillin V, ampicillin, amoxicillin, bacampicillin, cyclacillin, epicillin, hetacillin, pivampicillin, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, fiucloxacillin, carbenicillin, ticarcillin, avlocillin, mezlocillin, piperacillin, amdinocillin, cephalexin, cephradine, cefadoxil, cefaclor, cefazolin, cefuroxime axetil, cefamandole, cefonicid, cefoxitin, cefotaxime, ceftizoxime, cefinenoxine, ceftriaxone, moxalactam, cefotetan, ce
• a therapeutically active amount of a polypeptide may vary according to factors such as age, sex, medical complications (e.g., immunosuppressed conditions or diseases) and weight of the subject, and the ability of the antibody to elicit a desired response in the subject.
• the dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
• an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram body weight per day and more preferably from about 0.5 to 10, milligrams per kilogram body weight per day.
• the precise time of administration of the anti-PNAG antibody can be adjusted according to the patients needs.
• the anti-PNAG antibody can be given at any time prior to exposure to the biofilm-forming microbe (e,g, upon entry into hospital or at the start of a medical procedure).
• the anti-PNAG antibody can be administered between 0 and 240 hours prior to exposure to the biofilm-forming microbe, e.g., about 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9,10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 24, 30, 40, 60, 80, 90, 100, 110, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 2010, 220, 230, and/or 240 hours.
• S. aureus (ATCC 33592) was grown overnight in TSB medium, adjusted at 1E+07 CFU/ml. The bacteria were then aliquoted into MBEC plates (MBEC AssaysTM, Innovotech) containing 5, or 20 ug/ml of concentration of F598 or human IgG control and incubated for 5.5h. Following the incubation period, the pegs of the MBEC plates were removed, washed and the amount of formed biofilm quantified using by luminescence (BacTiter-GloTM
• F598 demonstrated a clear protective effect against S. aureus in vitro biofilm development. Specifically, F598 showed more than 50% inhibition of S. aureus biofilm development (43 +/- 8 % and 37+/- 3%, at 5 and 20 ⁇ g/ml, respectively) (see Figure 1).
• S. epidermidis (clinical isolate 1457) was grown in multiwell plates in TSB medium for 8 h in the presence of 25, 50, 100, or 200 ⁇ g/ml of F598 or human IgGl control. Wells were then washed and stained with crystal violet. Biofilms were quantified by measuring the sample absorbance at a wavelength of 490 nm.
• F598 demonstrated a clear, dose -dependent protective effect against S. epidermidis 1457 in vitro biofilm development. Specifically, at 25 ⁇ g/ml biofilm formation was 40% of the control (0.2 vs 0.5 OD), and at 200 ⁇ g/ml biofilm formation was 20% of the control (0.1 vs 0.5 OD) (see Figure 2).
• S. epidermidis (clinical isolate RP62A) was grown in multiwell plates in TSB medium for 8 h in the presence of 19, 38, or 76 ⁇ g/ml of F598 or PBS buffer control. Wells were then washed and stained with crystal violet. Biofilms were quantified by measuring the sample absorbance at a wavelength of 490 nm.
• F598 demonstrated a clear, dose-dependent protective effect against S. epidermidis RP62A in vitro biofilm development. Specifically, 19, 38, and 76 ⁇ g/ml of F598 reduced biofilm formation 36, 60 and 76%, respectively (relative to the corresponding PBS control) (see Figure 3).

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