US20080038193A1 - Antibodies for use in targeted and temporary treatment of humans and animals - Google Patents
Antibodies for use in targeted and temporary treatment of humans and animals Download PDFInfo
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- US20080038193A1 US20080038193A1 US11/973,033 US97303307A US2008038193A1 US 20080038193 A1 US20080038193 A1 US 20080038193A1 US 97303307 A US97303307 A US 97303307A US 2008038193 A1 US2008038193 A1 US 2008038193A1
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6815—Enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
Definitions
- the invention relates to antibodies or fragments thereof which can be used for “targeting” of (diagnostically, therapeutically and cosmetically) active substances to desired epitopes.
- the invention further relates to the use of these antibodies or fragments thereof in the targeted and temporary diagnostic, therapeutic and cosmetic treatment of externally accessible parts of the human and animal body.
- an antibody-antigen binding to localize, for instance, a therapeutically or diagnostically active substance on a determined epitope at a specific site of the body is already known per se.
- EP 0736544 are described antibodies which recognize specific protein structures on the tooth, whereby therapeutic or cosmetic substances can be applied specifically and locally to the tooth. Targeted and local administering of active substances achieves maximum effectiveness of these substances.
- such antibodies or fragments thereof can be used for the detection of, for instance, dental plaque.
- a dye for instance, erythrocin
- Early detection of plaque provides the possibility of removing the plaque before it causes infectious or carious disorders of the teeth and/or surrounding tissues.
- the plaque By bringing the teeth and surrounding tissues into contact with the antibody coupled to a dye which detects the plaque, the plaque can be made visible and then removed by cleaning the teeth and the surrounding tissues, in particular at those locations where the presence of plaque has been determined.
- a drawback of the use of antibodies or fragments thereof for detecting dental plaque via this method is the fact that the antibody/epitope binding is not easily broken, whereby dyes visible to the eye remain visible for a longer time. This can be avoided by using dyes based on fluoresceine which are only visible during irradiation with UV-light. These agents are, however, rather time-consuming in use and not easy for the consumer to use.
- the therapeutic treatment of infections in the oral cavity with, for instance, systemic antibiotics has the important drawback that the antibiotic spreads through the entire body and also reaches other organs and tissues, whereby the natural microbial flora of, for instance, the gastrointestinal tract can be disrupted.
- the use of antibiotic gel in the oral cavity may partially prevent this, but has the drawback that it does not remain properly adhered.
- antibodies or fragments thereof which bind to an epitope under specifically chosen conditions, and which elute from that epitope under specifically chosen different conditions.
- the antibodies can be removed at any desired moment.
- an infection in, for instance, the oral cavity can be treated very locally and in a targeted manner, and antibodies which are coupled to a dye and which are used for the detection of dental plaque or other oral pathogens can be removed at any desired moment, without lips, tongue and gums remaining colored for a long time.
- Phage-display is a technique for making human antibodies or antibody fragments which can serve for diagnostic or therapeutic purposes and/or for use in research.
- the phage-display technique makes use of the possibility of expressing proteins in the coat of bacteriophages.
- bacteriophages are transfected with human variable (V) genes or with a combination of human variable and constant genes which form the basis for the composition of human antibodies.
- V human variable
- g3p g3p-antibody fusion proteins appear after synthesis on the surface of the bacteriophage.
- phage libraries or banks of Fab fragments or single chain Fv fragments (scFv) with different antibody specificities.
- phage libraries are used in the present invention to select the antibody fragments with the specific property that these antibody fragments bind to a determined epitope under specifically chosen conditions and that the binding between antibody fragment and epitope is broken in specifically chosen different conditions.
- These specifically chosen conditions can, for instance, be formed by the pH or the ionic strength.
- these conditions preferably lie within physiologically acceptable limits.
- the pH can, for instance, be varied between 4 and 8.5 and the ionic strength between 0 and 13 M.
- FIGS. 1 a - c , 2 a - c , 3 a - c and 4 a - c show the DNA patterns of different clones from respective selection procedures A, B, C and D.
- DNA from the clones from the different selection procedures was replicated using PCR, cleaved with restriction-enzyme BstN-1 and subsequently analyzed on 3% agarose gel.
- Fab monoclonal phage-antibody fragments
- “Fragments” are understood to mean F(ab), F(ab)′, F(ab)′ 2 or scFv fragments.
- therapeutically or cosmetically active substances can be coupled to the obtained antibodies or fragments as is described in EP 0453097, EP 0451972 and EP 0450800, or as according to Lal et al. ( Immunol. Meth. 1985; 79: 307-318).
- Such substances can, for instance, be enzymes, dyes, fluorescent substances, radioactive substances or antimicrobial compounds.
- Enzymes can be used as antimicrobial agents (therapeutic), or as a bleaching agent for removing deposits on the teeth (cosmetic treatment).
- Dyes can be used to detect, for instance, dental plaque or other oral pathogenic microorganisms (diagnostic).
- Antimicrobial compounds can be used to treat infections in the oral cavity or other externally accessible parts of the body.
- Antibodies or fragments thereof according to the invention can be used for the treatment of externally accessible parts of the human body, such as the oral cavity, but also the skin and the gastrointestinal and urogenital system.
- An example of an application of antibodies according to the invention is bringing antimicrobial compounds into contact with species of the oral microflora.
- the oral microflora contains a wide diversity of microbial species including the pathogens: Actinomyces actinomycetem comitans, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Bacteroides forsythus, Eikenella corrodens, Treponema denticola, Campylobacter lectus , and Fusobacterium nucleatum . A number of these species make a significant contribution toward the forming of plaque.
- antimicrobial compound can, for instance, be used an enzyme such as an oxidase (for instance, glucose oxidase, lactase oxidase or uric acid oxidase), a peroxidase (for instance, horseradish peroxidase, myeloperoxidase, lactoperoxidase or chloroperoxidase), a protease (for instance, papain, pepsin, trypsin, ficin or bromelin), a cell wall-lysing enzyme (for instance, lysozyme), or a plaque matrix inhibitor (for instance, dextranase or mutanase).
- an enzyme such as an oxidase (for instance, glucose oxidase, lactase oxidase or uric acid oxidase), a peroxidase (for instance, horseradish peroxidase, myeloperoxidase, lactoperoxidase or
- the invention further relates to the use of such antibodies or antibody fragments for the manufacture of a medication for targeted and temporary diagnostic, therapeutic and cosmetic treatment of externally accessible parts of the human and animal body.
- a medication comprising the antibodies or fragments thereof according to the invention can take a number of forms, depending on the area of the body where the medication has to be applied.
- the medication can, for instance, take the form of a mouthwash, a lozenge or chewing tablet, a teeth cleaning agent, cream, ointment or chewing gum.
- Example 1 describes the selection of antibodies and fragments thereof, wherein a Fab-phage library is used as the starting point.
- a Fab-phage library can, for instance, be made as described in Marks et al. ( J. Mol. Biol. 222: 581-597, 1991) and as described in Haard et al. ( J. Biol. Chem. 274: 18218-18230, 1999).
- the monoclonal antibodies which recognize a specific epitope on the bacterium Staphylococcus epidermidis ( S. epidermidis ) and which bind reversibly under conditions which can be used in the oral cavity were obtained using a human Fab phage library.
- the infected bacteria were centrifuged and resuspended in culture buffer. After an overnight culture, the bacteria were removed using centrifugation, and the phages were precipitated out of the culture medium by means of PEG precipitation. The precipitated phages were resuspended in one of the four binding buffers of Table 1, and were subsequently used in the different selection procedures.
- Table 2 shows the titration results of the different selection procedures on S. epidermidis . These results demonstrate that for selection procedure B and C, the number of specific phages does not increase any further after three selection rounds. In addition, the fact that in selection B, C, and D the titer of the final washing step is lower than the titer of the eluted fraction (output) shows that specific elution has occurred. In selection A, this is not the case and there is, therefore, probably no specific elution in this case. The increasing Output/Input ratio shows the enrichment of binding and eluting phages after the different selection rounds.
- 188 clones (24 clones from round 3 and 23 clones from round 4 of selection A and D, and 42 clones from round 3 and 5 clones from round 4 of selection B and C) were selected at random and tested as phage-antibodies in the Whole Cell ELISA in a 96-well plate, as described below, for screening for antibodies which bind specifically to S. epidermidis .
- Monoclonal phage particles (individual clones) were incubated with S. epidermidis (2*10 7 bacteria per well) in binding buffer in a 96-well plate. The bacteria were then washed thoroughly with binding buffer (by means of centrifuging followed by resuspension), afterwards the phage particles were eluted with elution buffer (or binding buffer as control) for 30 minutes. After the elution, the bacteria were centrifuged and the presence of phage particles on the bacteria was demonstrated by means of peroxidase-labeled mouse-anti-M13 antibodies and 3,3′,5,5′-tetramethylbenzidine (TMB). The plate was measured at 405 nm. In the Whole Cell ELISA, the same combinations of binding and elution buffers were used as during the selection.
- Clones with an ELISA signal that was twice as high as the background signal when elution took place with the binding buffer (control) were designated “positive clones.” Positive clones are, therefore, all clones which bind specifically to the bacterium at the chosen binding condition, irrespective of whether they elute in other conditions.
- Clones with an ELISA signal after elution with binding buffer which was twice as high as the ELISA signal after elution with elution buffer were designated “specifically eluting clones.”
- the specifically eluting clones are, therefore, the antibody-phage clones which bind in the binding buffer used, and which do not elute in the binding buffer but do so when washed with the elution buffer.
- the specifically eluting clones therefore, bind under specifically chosen conditions and elute under specifically chosen different conditions. Table 3 shows the results of the Whole Cell ELISA.
- DNA of the individual clones was replicated using PCR and then cleaved with restriction-enzyme BstN-1.
- the DNA samples were analyzed on 3% agarose gel with 0.5 ⁇ g/ml ethidium bromide.
- FIGS. 1 a - c , 2 a - c , 3 a - c and 4 a - c are the DNA patterns of the individual clones.
- the clones are numbered in accordance with their position on the ELISA plate. The results show that respectively 2, 12, 13 and 32 different DNA patterns (“fingerprints”) are present in selections A, B, C and D. Patterns 4, 5 and 14 occur in both selections B and D.
- Patterns 10 and 12 are found in both selections B and C. Pattern 24 occurs in selections C and D and pattern 3 is found in selections B, C and D. There are, therefore, clones which elute due to a pH-shock, and there are other clones which elute due to a salt-shock, or through a combination of pH and salt-shock.
- the 16 specific clones according to the invention were tested in an ELISA for binding to streptavidin, biotin-labeled BSA, thyroglobulin and to an empty well. None of the 16 clones bound to any of the coated antigens.
- the binding to S. epidermidis in elution buffer and to S. epidermidis in binding buffer was tested in the Whole Cell ELISA. All 16 clones according to the invention bound only to S. epidermidis in binding buffer and not in elution buffer.
- a clone from selection C (Example 1) was used to prepare a toothpaste with phage-antibody fragments coupled to erythrocin for the purpose of demonstrating the presence of S. epidermidis in the oral cavity.
- the phage-antibody fragments from selection procedure C bind to S. epidermidis at a pH of 8.5 and a salt concentration of 1 M NaCl.
- the binding of antibody to S. epidermidis is made visible by the bound dye.
- pH 7 water
- An ionic strength of between about 0 and 13 M can be used in the oral cavity.
- the antibody or a fragment thereof having coupled thereto a suitable diagnostically, therapeutically or cosmetically active substance binds at an ionic strength of between 0 and 500 mM. The binding is then broken at any desired moment by rinsing with a mouthwash having an ionic strength of 1-13 M.
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Abstract
Described is an antibody, or a fragment of an antibody, which binds to an epitope under specifically chosen conditions, and the binding of which to that epitope is broken under specifically chosen different conditions, in addition to an antibody or fragment thereof with which a diagnostically, therapeutically or cosmetically active substance is optionally conjugated, for targeted and temporary diagnostic, therapeutic or cosmetic treatment of externally accessible parts of the human and animal body, wherein both the specifically chosen binding conditions and the conditions wherein the binding is broken lie within physiologically acceptable limits and are formed by the pH and/or ionic strength. The invention further relates to the use of such an antibody or fragment thereof for manufacturing a medication for targeted and temporary treatment of externally accessible parts of the human and animal body, in addition to a composition comprising at least one antibody, or a fragment thereof, according to the invention such as a teeth cleaning agent, mouthwash, mouth spray, chewing tablet, cream or ointment.
Description
- This application is a continuation of co-pending U.S. patent application Ser. No. 09/780,205, filed Feb. 9,2001, now U.S. Pat. No. ______, which application claims priority to, and is a continuation of, International Application No. PCT/NL99/00509, filed on Aug. 10, 1999, designating the United States of America, which PCT International Patent Application itself claims priority from the Netherlands Application Serial No. 1009834, filed Aug. 10, 1998, the contents of each of which are incorporated herein by this reference.
- The invention relates to antibodies or fragments thereof which can be used for “targeting” of (diagnostically, therapeutically and cosmetically) active substances to desired epitopes. The invention further relates to the use of these antibodies or fragments thereof in the targeted and temporary diagnostic, therapeutic and cosmetic treatment of externally accessible parts of the human and animal body.
- The use of an antibody-antigen binding to localize, for instance, a therapeutically or diagnostically active substance on a determined epitope at a specific site of the body is already known per se. Described, for instance, in U.S. Pat. No. 5,490,988 and WO 95/01155 are antibodies and fragments of antibodies to which a therapeutically active substance is coupled for the “targeting” of antimicrobial substances to epitopes of pathogenic compounds in the oral cavity. In EP 0736544 are described antibodies which recognize specific protein structures on the tooth, whereby therapeutic or cosmetic substances can be applied specifically and locally to the tooth. Targeted and local administering of active substances achieves maximum effectiveness of these substances.
- Through conjugation with a dye (for instance, erythrocin), such antibodies or fragments thereof can be used for the detection of, for instance, dental plaque. Early detection of plaque provides the possibility of removing the plaque before it causes infectious or carious disorders of the teeth and/or surrounding tissues. By bringing the teeth and surrounding tissues into contact with the antibody coupled to a dye which detects the plaque, the plaque can be made visible and then removed by cleaning the teeth and the surrounding tissues, in particular at those locations where the presence of plaque has been determined.
- A drawback of the use of antibodies or fragments thereof for detecting dental plaque via this method is the fact that the antibody/epitope binding is not easily broken, whereby dyes visible to the eye remain visible for a longer time. This can be avoided by using dyes based on fluoresceine which are only visible during irradiation with UV-light. These agents are, however, rather time-consuming in use and not easy for the consumer to use.
- The therapeutic treatment of infections in the oral cavity with, for instance, systemic antibiotics has the important drawback that the antibiotic spreads through the entire body and also reaches other organs and tissues, whereby the natural microbial flora of, for instance, the gastrointestinal tract can be disrupted. The use of antibiotic gel in the oral cavity may partially prevent this, but has the drawback that it does not remain properly adhered.
- Provided are antibodies or fragments thereof which bind to an epitope under specifically chosen conditions, and which elute from that epitope under specifically chosen different conditions. By changing the conditions such that the binding between epitope and antibody is broken, the antibodies can be removed at any desired moment. In this way, an infection in, for instance, the oral cavity can be treated very locally and in a targeted manner, and antibodies which are coupled to a dye and which are used for the detection of dental plaque or other oral pathogens can be removed at any desired moment, without lips, tongue and gums remaining colored for a long time.
- An antibody is selected using the per se known “phage-display” technique. Phage-display is a technique for making human antibodies or antibody fragments which can serve for diagnostic or therapeutic purposes and/or for use in research. The phage-display technique makes use of the possibility of expressing proteins in the coat of bacteriophages. In this technique, bacteriophages are transfected with human variable (V) genes or with a combination of human variable and constant genes which form the basis for the composition of human antibodies. By ensuring that the synthesized antibodies are expressed as fusion proteins with a bacteriophagic coat protein (for instance, g3p), g3p-antibody fusion proteins appear after synthesis on the surface of the bacteriophage.
- There are large so-called phage libraries (or banks) of Fab fragments or single chain Fv fragments (scFv) with different antibody specificities. Such phage libraries are used in the present invention to select the antibody fragments with the specific property that these antibody fragments bind to a determined epitope under specifically chosen conditions and that the binding between antibody fragment and epitope is broken in specifically chosen different conditions.
- These specifically chosen conditions can, for instance, be formed by the pH or the ionic strength. For application in the treatment of the human and animal body, these conditions preferably lie within physiologically acceptable limits. For use in the oral cavity, the pH can, for instance, be varied between 4 and 8.5 and the ionic strength between 0 and 13 M.
-
FIGS. 1 a-c, 2 a-c, 3 a-c and 4 a-c show the DNA patterns of different clones from respective selection procedures A, B, C and D. For this purpose, DNA from the clones from the different selection procedures was replicated using PCR, cleaved with restriction-enzyme BstN-1 and subsequently analyzed on 3% agarose gel. - Using a selection procedure as described in Example 1, monoclonal phage-antibody fragments (Fab) are obtained with the properties desired for the application such as affinity, specificity and pH and/or ionic strength sensitivity. By optionally changing the expression system, it is ultimately possible to bring about that Fab fragments or even intact human monoclonal antibodies are secreted into the culture medium. These antibody fragments or intact antibodies can then be isolated easily from the culture medium.
- “Fragments” are understood to mean F(ab), F(ab)′, F(ab)′2 or scFv fragments.
- Diagnostically, therapeutically or cosmetically active substances can be coupled to the obtained antibodies or fragments as is described in EP 0453097, EP 0451972 and EP 0450800, or as according to Lal et al. (Immunol. Meth. 1985; 79: 307-318).
- Such substances can, for instance, be enzymes, dyes, fluorescent substances, radioactive substances or antimicrobial compounds. Enzymes can be used as antimicrobial agents (therapeutic), or as a bleaching agent for removing deposits on the teeth (cosmetic treatment). Dyes can be used to detect, for instance, dental plaque or other oral pathogenic microorganisms (diagnostic). Antimicrobial compounds can be used to treat infections in the oral cavity or other externally accessible parts of the body.
- Antibodies or fragments thereof according to the invention can be used for the treatment of externally accessible parts of the human body, such as the oral cavity, but also the skin and the gastrointestinal and urogenital system.
- An example of an application of antibodies according to the invention is bringing antimicrobial compounds into contact with species of the oral microflora. The oral microflora contains a wide diversity of microbial species including the pathogens: Actinomyces actinomycetem comitans, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Bacteroides forsythus, Eikenella corrodens, Treponema denticola, Campylobacter lectus, and Fusobacterium nucleatum. A number of these species make a significant contribution toward the forming of plaque. By coupling antimicrobial substances to antibodies or fragments thereof which are targeted against epitopes on these pathogenic compounds, these species can be “attacked” very locally and in a targeted manner. As antimicrobial compound can, for instance, be used an enzyme such as an oxidase (for instance, glucose oxidase, lactase oxidase or uric acid oxidase), a peroxidase (for instance, horseradish peroxidase, myeloperoxidase, lactoperoxidase or chloroperoxidase), a protease (for instance, papain, pepsin, trypsin, ficin or bromelin), a cell wall-lysing enzyme (for instance, lysozyme), or a plaque matrix inhibitor (for instance, dextranase or mutanase).
- The invention further relates to the use of such antibodies or antibody fragments for the manufacture of a medication for targeted and temporary diagnostic, therapeutic and cosmetic treatment of externally accessible parts of the human and animal body. A medication comprising the antibodies or fragments thereof according to the invention can take a number of forms, depending on the area of the body where the medication has to be applied. For application in the oral cavity, the medication can, for instance, take the form of a mouthwash, a lozenge or chewing tablet, a teeth cleaning agent, cream, ointment or chewing gum.
- The invention is further illustrated using the annexed examples and figures. Example 1 describes the selection of antibodies and fragments thereof, wherein a Fab-phage library is used as the starting point. A Fab-phage library can, for instance, be made as described in Marks et al. (J. Mol. Biol. 222: 581-597, 1991) and as described in Haard et al. (J. Biol. Chem. 274: 18218-18230, 1999).
- The Examples and figures serve are illustrative and are in no way limiting for the invention.
- Selection of Antibodies
- The monoclonal antibodies which recognize a specific epitope on the bacterium Staphylococcus epidermidis (S. epidermidis) and which bind reversibly under conditions which can be used in the oral cavity were obtained using a human Fab phage library.
- 1.1 Production of Phage Particles From a Library of Antibodies Cloned in Phagemids in E. coli
- The E. coli bacteria were cultured to an OD600 nm of 0.5 (=2*105 bacteria) and helper phages were then added to bring about the formation of phage particles. The infected bacteria were centrifuged and resuspended in culture buffer. After an overnight culture, the bacteria were removed using centrifugation, and the phages were precipitated out of the culture medium by means of PEG precipitation. The precipitated phages were resuspended in one of the four binding buffers of Table 1, and were subsequently used in the different selection procedures.
- 1.2 Selection of the Phages
- 1*109 bacteria (S. epidermidis) (in the different binding buffers, see table 1) were incubated with phages (input) (see 1.1). The bacteria were then washed 12 times with binding buffer and the phages that had bound were eluted with the correct elution buffer (see table 1). After elution, 55% of the bacteria was still alive.
- The eluted phages were replicated via infection of E. coli, followed by culture of the infected E. coli according to 1.1. The cultured eluted phages were then reused in the following selection round (input). The described selection procedures were carried out four times.
TABLE 1 Selection Binding buffer Elution buffer A PBS (pH 7.4) pH 4.5 B PBS (pH 7.4) pH 4.5 + 1 M NaCl C pH 8.5 + 1 M NaCl Milli Q D pH 8.5 pH 4.5 + 1 M NaCl - Table 2 shows the titration results of the different selection procedures on S. epidermidis. These results demonstrate that for selection procedure B and C, the number of specific phages does not increase any further after three selection rounds. In addition, the fact that in selection B, C, and D the titer of the final washing step is lower than the titer of the eluted fraction (output) shows that specific elution has occurred. In selection A, this is not the case and there is, therefore, probably no specific elution in this case. The increasing Output/Input ratio shows the enrichment of binding and eluting phages after the different selection rounds.
- 1.3 Screening Using “Whole Cell” ELISA
- 188 clones (24 clones from
round 3 and 23 clones fromround 4 of selection A and D, and 42 clones fromround round 4 of selection B and C) were selected at random and tested as phage-antibodies in the Whole Cell ELISA in a 96-well plate, as described below, for screening for antibodies which bind specifically to S. epidermidis.TABLE 2 Selection Round Input1 Titer2 Output3 Output/ Input A 1 6.9*1012 — 7.5*104 1.1*10−8 2 7.4*1013 — 1.7*105 2.3*10−9 3 1.8*1013 1.1*108 1.3*107 7.2*10−7 4 2.5*1013 8.8*108 2.0*108 8.0*10−6 B 1 5.4*1012 — 5.8*105 1.1*10−7 2 8.3*1013 — 6.6*107 8.0*10−7 3 8.3*1012 5.5*109 3.4*1010 4.1*10−3 4 5.8*1012 6.5*109 3.9*1010 6.7*10−3 C 1 6.0*1012 — 1.2*106 2.0*10−7 2 2.7*1013 — 2.0*107 7.4*10−7 3 1.1*1013 3.1*109 3.4*1010 3.1*10−3 4 4.7*1012 2.1*109 3.1*1010 6.6*10−3 D 1 6.5*1012 — 1.9*107 2.9*10−6 2 3.6*1013 — 1.1*108 3.1*10−6 3 1.9*1013 1.7*108 5.0*109 2.6*10−4 4 9.3*1012 4.9*108 1.1*1010 1.2*10−3
1Input (total cfu) (cfu = colony forming unit)
2Titer (total cfu of the final washing step before elution)
3Output (total cfu)
- Monoclonal phage particles (individual clones) were incubated with S. epidermidis (2*107 bacteria per well) in binding buffer in a 96-well plate. The bacteria were then washed thoroughly with binding buffer (by means of centrifuging followed by resuspension), afterwards the phage particles were eluted with elution buffer (or binding buffer as control) for 30 minutes. After the elution, the bacteria were centrifuged and the presence of phage particles on the bacteria was demonstrated by means of peroxidase-labeled mouse-anti-M13 antibodies and 3,3′,5,5′-tetramethylbenzidine (TMB). The plate was measured at 405 nm. In the Whole Cell ELISA, the same combinations of binding and elution buffers were used as during the selection.
- Clones with an ELISA signal that was twice as high as the background signal when elution took place with the binding buffer (control) were designated “positive clones.” Positive clones are, therefore, all clones which bind specifically to the bacterium at the chosen binding condition, irrespective of whether they elute in other conditions. Clones with an ELISA signal after elution with binding buffer which was twice as high as the ELISA signal after elution with elution buffer were designated “specifically eluting clones.” The specifically eluting clones are, therefore, the antibody-phage clones which bind in the binding buffer used, and which do not elute in the binding buffer but do so when washed with the elution buffer. The specifically eluting clones, therefore, bind under specifically chosen conditions and elute under specifically chosen different conditions. Table 3 shows the results of the Whole Cell ELISA.
- The fact that in the group of clones from selection A not a single specifically eluting clone was found confirms the supposition that in selection A, specific elution did not occur (see 1.2).
TABLE 3 number of specifically Selection number of positive clones eluting clones A 47/47 0/47 B 47/47 30/47 C 47/47 7/47 D 25/47 13/47
1.4 “Fingerprint Analysis” of the Positive Clones - All 188 clones from the four selections were screened with fingerprint analysis as described hereinbelow in order to determine the diversity of the clones. Using the fingerprint analysis, DNA patterns were obtained which were subsequently used to distinguish the different clones. The clones were tested for the presence of an insert of 1700 bp (clones which code for Fab must have an insert of about 1700 bp). All tested clones (except one clone from selection C, and eight clones from selection D) contained the specific insert.
- For the fingerprint analysis, DNA of the individual clones was replicated using PCR and then cleaved with restriction-enzyme BstN-1. The DNA samples were analyzed on 3% agarose gel with 0.5 μg/ml ethidium bromide. Shown in
FIGS. 1 a-c, 2 a-c, 3 a-c and 4 a-c are the DNA patterns of the individual clones. The clones are numbered in accordance with their position on the ELISA plate. The results show that respectively 2, 12, 13 and 32 different DNA patterns (“fingerprints”) are present in selections A, B, C andD. Patterns D. Patterns C. Pattern 24 occurs in selections C and D andpattern 3 is found in selections B, C and D. There are, therefore, clones which elute due to a pH-shock, and there are other clones which elute due to a salt-shock, or through a combination of pH and salt-shock. - When the results of the Whole Cell ELISA are compared to the results of the DNA patterns (see table 4), there are 16 clones (with different DNA patterns) which are positive in the binding to S. epidermidis and which elute specifically after binding.
TABLE 4 fingerprint pattern selection 6, 7, 8, 9, 11, 13 B 17, 18, 21, 22 C 47, 51 D 12 B, C 5, 14 B, D 3 B, C, D
1.5 Determining the Specificity - The 16 specific clones according to the invention were tested in an ELISA for binding to streptavidin, biotin-labeled BSA, thyroglobulin and to an empty well. None of the 16 clones bound to any of the coated antigens. The binding to S. epidermidis in elution buffer and to S. epidermidis in binding buffer was tested in the Whole Cell ELISA. All 16 clones according to the invention bound only to S. epidermidis in binding buffer and not in elution buffer.
- Toothpaste
- A clone from selection C (Example 1) was used to prepare a toothpaste with phage-antibody fragments coupled to erythrocin for the purpose of demonstrating the presence of S. epidermidis in the oral cavity. The phage-antibody fragments from selection procedure C bind to S. epidermidis at a pH of 8.5 and a salt concentration of 1 M NaCl. The binding of antibody to S. epidermidis is made visible by the bound dye. By then rinsing with water (pH 7), the binding is broken and the bound erythrocin removed.
- Ionic Strength as Specifically Chosen Condition
- An ionic strength of between about 0 and 13 M can be used in the oral cavity.
- In a different embodiment according to the invention, the antibody or a fragment thereof having coupled thereto a suitable diagnostically, therapeutically or cosmetically active substance binds at an ionic strength of between 0 and 500 mM. The binding is then broken at any desired moment by rinsing with a mouthwash having an ionic strength of 1-13 M.
Claims (32)
1. An antibody, or fragment thereof, which binds to an epitope under specifically chosen conditions, the bond of the antibody, or fragment thereof, to the epitope being broken under specifically chosen different conditions, wherein both the specifically chosen binding conditions and the conditions under which the bond is broken lie within physiologically acceptable limits.
2. The antibody, or fragment thereof, of claim 1 further comprising a diagnostically, therapeutically or cosmetically active substance conjugated thereto.
3. The antibody, or fragment thereof, of claim 1 , wherein the specifically chosen binding conditions and the conditions under which the bond is broken are dependent upon pH.
4. The antibody, or fragment thereof, of claim 3 , wherein the antibody, or fragment thereof, binds to the epitope at a pH within a range of 4 and 8.5, and the bond between the antibody, or fragment thereof, and the epitope is broken at a different pH within the range of 4 and 8.5.
5. The antibody, or fragment thereof, of claim 3 , wherein the antibody, or fragment thereof, binds to the epitope at a pH within a range chosen from a range of between 4 and 7 and a range of between 7 and 8.5, and the bond between the antibody, or fragment thereof, and the epitope is broken at a pH of 7.
6. The antibody, or fragment thereof, of claim 3 , wherein the antibody, or fragment thereof, binds to the epitope at a pH of 7, and the bond between the antibody, or fragment thereof, and the epitope is broken at a pH within a range chosen from a range of between 4 and 7 and a range of between 7 and 8.5.
7. The antibody, or fragment thereof, of claim 3 , wherein the antibody, or fragment thereof, binds to the epitope at a pH within a range chosen from a range of between 4 and 6 and a range of between 8 and 8.5, and the bond between the antibody, or fragment thereof, and the epitope is broken at a pH of between 6 and 8.
8. The antibody, or fragment thereof, of claim 2 , wherein the antibody, or fragment thereof, binds to the epitope at a pH of between 6 and 8, and the bond between the antibody, or fragment thereof, and the epitope is broken at a pH within a range chosen from a range of between 4 and 6 and a range of between 8 and 8.5.
9. The antibody, or fragment thereof, of claim 1 , wherein the specifically chosen binding conditions and the conditions under which the bond is broken are dependent upon ionic strength.
10. The antibody, or fragment thereof, of claim 9 , wherein the antibody, or fragment thereof, binds to the epitope at an ionic strength within a range of 0 and 13 M, and the bond between the antibody, or fragment thereof, and the epitope is broken at a different ionic strength within the range of 0 and 13 M.
11. The antibody, or fragment thereof, of claim 9 , wherein the antibody, or fragment thereof, binds to the epitope at an ionic strength within a range of 0 and 500 mM, and the bond between the antibody, or fragment thereof, and the epitope is broken at an ionic strength within the range of 1 and 13 M.
12. The antibody, or fragment thereof, of claim 9 , wherein the antibody, or fragment thereof, binds the epitope at a pH within a range of 4 and 8.5 and/or at an ion concentration within a range of 0 and 13 M, and the bond between the antibody, or fragment thereof, and the epitope is broken at a different pH within the range of 4 and 8.5 and/or a different ionic strength within the range of 0 and 13 M.
13. The antibody, or fragment thereof, of claim 1 , wherein the antibody, or fragment thereof, comprises a F(ab), F(ab)′, F(ab)′2 or an scFv.
14. A method of using the antibody, or fragment thereof, of claim 1 , as a targeted or temporary diagnostic, therapeutic or cosmetic treatment of externally accessible parts of a subject's body.
15. The method according to claim 14 , wherein said targeted or temporary diagnostic, therapeutic or cosmetic treatment comprises a treatment of an oral cavity of a human or an animal body.
16. The method according to claim 15 , wherein said antibody, or fragment thereof, is capable of bleaching teeth included in said oral cavity.
17. The method according to claim 15 , wherein said antibody, or fragment thereof, is able to detect plaque in an oral cavity.
18. The method according to claim 15 , wherein said antibody, or fragment thereof, is capable of removing plaque in said oral cavity.
19. The method according to claim 14 , wherein said targeted or temporary diagnostic, therapeutic or cosmetic treatment comprises a treatment for fighting infections in externally accessible parts of a human or an animal body.
20. The antibody, or fragment thereof, of claim 2 , wherein the diagnostically, therapeutically or cosmetically active substance comprises an enzyme.
21. The antibody, or fragment thereof, of claim 20 , wherein the enzyme is selected from the group consisting of an oxidase, a peroxidase, a protease, a cell-wall lysing enzyme, and a plaque matrix inhibitor.
22. The antibody, or fragment thereof, of claim 21 , wherein the enzyme comprises an oxidase selected from the group consisting of glucose oxidase, lactase oxidase, and uric acid oxidase.
23. The antibody, or fragment thereof, of claim 21 , wherein the enzyme comprises a peroxidase selected from the group consisting of horseradish peroxidase, myeloperoxidase, lactoperoxidase, and chloroperoxidase.
24. The antibody, or fragment thereof, of claim 21 , wherein the enzyme comprises a protease selected from the group consisting of papain, pepsin, trypsin, ficin, and bromelin.
25. The antibody, or fragment thereof, of claim 21 , wherein the enzyme comprises lysozyme.
26. The antibody, or fragment thereof, of claim 21 , wherein the enzyme comprises a plaque matrix inhibitor selected from the group consisting of dextranase and mutanase.
27. The antibody, or fragment thereof, of claim 2 , wherein the diagnostically, therapeutically or cosmetically active substance comprises a fluorescent or radioactive substance.
28. The antibody, or fragment thereof, of claim 2 , wherein the antibody or fragment thereof is capable of binding an epitope of a pathogenic microorganism or other pathogenic compound.
29. The antibody, or fragment thereof, of claim 28 , wherein said pathogenic microorganism is selected from the group consisting of Actinomyces actinomycetem comitans, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus mutans, Bacteroides forsythus, Eikenella corrodens, Treponema denticola, Campylobacter lectus, and Fusobacterium nucleatum.
30. A composition comprising:
at least one antibody, or fragment thereof, as claimed in claim 1; and
at least one physiologically acceptable diluent, solvent or carrier.
31. (canceled)
32. (canceled)
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US11/973,033 US20080038193A1 (en) | 1998-08-10 | 2007-10-05 | Antibodies for use in targeted and temporary treatment of humans and animals |
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Cited By (2)
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US20160324939A1 (en) * | 2014-06-09 | 2016-11-10 | Ucl Business Plc | Anti-microbial agents and uses therefor |
WO2017165320A1 (en) * | 2016-03-20 | 2017-09-28 | The Trustees Of The University Of Pennsylvania | Codon optimization and ribosome profiling for increasing transgene expression in chloroplasts of higher plants |
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NL1009834C2 (en) | 1998-08-10 | 2000-02-11 | Stanislaus Laurens Johan Woute | Antibodies for use in the targeted and temporary treatment of humans and animals. |
US7611709B2 (en) * | 2004-05-10 | 2009-11-03 | Boehringer Ingelheim Pharma Gmbh And Co. Kg | 1,4 O-linked saccharose derivatives for stabilization of antibodies or antibody derivatives |
US7723306B2 (en) | 2004-05-10 | 2010-05-25 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Spray-dried powder comprising at least one 1,4 O-linked saccharose-derivative and methods for their preparation |
US7727962B2 (en) | 2004-05-10 | 2010-06-01 | Boehringer Ingelheim Pharma Gmbh & Co. Kg | Powder comprising new compositions of oligosaccharides and methods for their preparation |
US9526803B2 (en) | 2012-03-08 | 2016-12-27 | Julius-Maximilians-Universitaet Wuerzburg | Diagnostic chewing gum for pathogens |
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US4138476A (en) * | 1977-08-03 | 1979-02-06 | The United States Of America As Represented By The Secretary Of The Navy | Plaque dispersing enzymes as oral therapeutic agents by molecular alteration |
US5490988A (en) * | 1990-10-05 | 1996-02-13 | Chesebrough-Pond's Usa Co., Division Of Conopco, Inc. | Delivery of therapeutic agents to a target site |
US5571511A (en) * | 1990-10-22 | 1996-11-05 | The U.S. Government | Broadly reactive opsonic antibodies that react with common staphylococcal antigens |
US7288247B2 (en) * | 1998-08-10 | 2007-10-30 | Wouters Stanislaus Laurens Joh | Antibodies for use in targeted and temporary treatment of humans and animals |
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GB8528761D0 (en) * | 1985-11-22 | 1985-12-24 | Axon Healthcare Ltd | Enzyme-coupled antibodies |
AU641736B2 (en) * | 1990-03-21 | 1993-09-30 | Quest International B.V. | Delivery of agents |
DE69523440T2 (en) * | 1995-04-05 | 2002-05-29 | Unilever N.V., Rotterdam | Oral care products |
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- 1999-08-10 AU AU53882/99A patent/AU5388299A/en not_active Abandoned
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2007
- 2007-10-05 US US11/973,033 patent/US20080038193A1/en not_active Abandoned
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US4138476A (en) * | 1977-08-03 | 1979-02-06 | The United States Of America As Represented By The Secretary Of The Navy | Plaque dispersing enzymes as oral therapeutic agents by molecular alteration |
US5490988A (en) * | 1990-10-05 | 1996-02-13 | Chesebrough-Pond's Usa Co., Division Of Conopco, Inc. | Delivery of therapeutic agents to a target site |
US5571511A (en) * | 1990-10-22 | 1996-11-05 | The U.S. Government | Broadly reactive opsonic antibodies that react with common staphylococcal antigens |
US7288247B2 (en) * | 1998-08-10 | 2007-10-30 | Wouters Stanislaus Laurens Joh | Antibodies for use in targeted and temporary treatment of humans and animals |
Cited By (3)
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US20160324939A1 (en) * | 2014-06-09 | 2016-11-10 | Ucl Business Plc | Anti-microbial agents and uses therefor |
WO2017165320A1 (en) * | 2016-03-20 | 2017-09-28 | The Trustees Of The University Of Pennsylvania | Codon optimization and ribosome profiling for increasing transgene expression in chloroplasts of higher plants |
US11332754B2 (en) | 2016-03-20 | 2022-05-17 | The Trustees Of The University Of Pennsylvania | Codon optimization for increasing transgene expression in chloroplasts of higher seed plants |
Also Published As
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EP1104308A1 (en) | 2001-06-06 |
AU5388299A (en) | 2000-03-06 |
CA2340116A1 (en) | 2000-02-24 |
US20020034508A1 (en) | 2002-03-21 |
US7288247B2 (en) | 2007-10-30 |
NL1009834C2 (en) | 2000-02-11 |
WO2000009164A1 (en) | 2000-02-24 |
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