Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
  • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
  • C07K16/085—Herpetoviridae, e.g. pseudorabies virus, Epstein-Barr virus
  • C07K16/087—Herpes simplex virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• Herpes species have been made among the Herpes species. Some of the representative members include Herpes . zoster. Herpes simplex, and Herpes hominis (inc. Types I and II) .
• the enzyme-linked immunoassay procedure EIA
• the enzyme-linked monoclonal antibody can then be used in the known enzyme-linked immunosor- bent assay procedure to determine the presence of an antigenic substance.
• the serotype of the infecting organism can be determined, and appropriate treatment can then be initiated to rapidly and efficiently eliminate the disease.
• the present invention provides novel mono ⁇ clonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Herpes antigens and/or organisms.
• the invention also comprises labeled mono ⁇ clonal antibodies for use in diagnosing the presence of the Herpes antigens, each comprising a monoclonal antibody against one of the above- mentioned antigens to Herpes or to a particular species thereof and linked thereto an appro ⁇ priate label.
• the label can be chosen from the group consisting of a radioactive isotope, enzyme, fluorescent compound, chemilumines- cent compound, bioluminescent compound, ferromag ⁇ netic atom, or particle, or any other label.
• the invention is also directed to a therapeutic composition
• a therapeutic composition comprising a mono ⁇ clonal antibody for an antigen of Herpes and a carrier or diluent, as well as kits contain ⁇ ing at least one labeled monoclonal antibody to an antigen of a Herpes.
• the immunized spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e., mice, rats, and rabbits), bovine, ovine, canine, or the like, but the present invention will be described in connection with mice.
• the mouse is first immunized by injection of the particular Herpes antigen chosen generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody produc- tion against the antigen, as determined by conven ⁇ tional assay, it is given a booster injection of the appropriate Herpes antigen, and then killed so that the immunized spleen may be remov ⁇ ed. The fusion can then be carried out utilizing immunized spleen cells and an appropriate myeloma cell line.
• Amounts of antibody sufficient for labeling and subsequent commercial production are produced by the known techniques, such as by batch or continuous tissue culture or culture in vivo in mammals, such as mice.
• the monoclonal antibodies may be labeled with a multitude of different labels, such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
• labels such as enzymes, fluorescent compounds, luminescent compounds, radioactive compounds, ferromagnetic labels, and the like.
• the present invention will be described with reference to the use of an enzyme labeled monoclonal antibody.
• Some of the enzymes utilized as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase, or urease, and the like.
• Such linkage with enzymes can be accomplished by any one of the conventional and known methods, such as the Staphylococcal Protein A method, the glutaraldehyde method, the benzoquinone method, or the periodate method.
• a nonlabeled antigen and a specific antibody are combined with identical fluorescently labeled antigen. Both labeled and unlabeled antigen compete for antibody binding sites. The amount of labeled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of nonlabeled antigen.
• Examples of this particular type of fluorescent- i munoassay would include heterogenous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate Labeled Fluorescent Immunoassay. The most suit- able fluorescent probe, and the one most widely used is fluorescein. While fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimized for the probe utilized in the particular assay and in which the effect of scattering can be minimized.
• Fluorescence polarization In fluorescence polarization, a labeled sample is excited with polarized light and the degree of polarization of the emitted light is measured. As the antigen binds to the antibody its rotation slows down and the degree of polari- zation increases. Fluorescence polarization is simple, quick, and precise. However, at the present time its sensitivity is limited to the micromole per liter range and upper nano- mole per liter range with respect to antigens in biological samples.
• a further aspect of the present invention is a therapeutic composition
• a therapeutic composition comprising one or more of the monoclonal antibodies to the particular Herpes antigen or species, as well as a pharmacologically acceptable carrier or diluent.
• Such compositions can be used to treat humans and/or animals afflicted with some form of Herpes infections and they are used in amounts effective to cure; an amount which will vary widely dependent upon the individual being treated and the severity of the infection.
• One or more of the monoclonal antibodies can be assembled into a diagnostic kit for use in diagnosing for the presence of an antigen, antigens, or species of Herpes in various speci ⁇ mens.
• a rapid diagnostic method requiring limited technical skill could be widely used to screen pregnant mothers and all persons with genital lesions, as well as infants suspected of neonatal infections.
• the broadly cross-reactive monoclonal antibody which can identify the genus Herpes alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Herpes and/or other bacteria.
• kits In the past there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens with antiserum.
• the use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis.
• a rapid and precise kit could replace or augment existing tests and permit early direct therapy using precise antibiotics. Avoiding multiple antibiotics or more expensive or hazardous antibiotics would represent substantial patient and hospital sav ⁇ ings.
• a kit can be used on an out-patient basis. At present the lack of a rapid test giving "same day" answers may delay the initiation of treatment until the patient has developed more severe symptoms or may require the initiation of more costly therapy in a sick patient. A test that would return results within an hour or two would be a substantial convenience to patients.
• kit could be included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Herpes.
• kits comprising at least one labeled monoclonal antibody against a particular Herpes antigen or species, as well as any appropriate stains, counterstains , or reagents.
• Specific antigens to be detected in this kit include the antigens of Herpes hominus (specifically Type II, which applicant has further divided into four subgroups) ; Herpes simplex; and Herpes zoster.
• FCS Foetal Calf Serum
• PBS phosphate-buffered saline pfu - plaque-forming units
• % T refers to vaccine concentration measured in a 1 cm light path
• Monoclonal antibodies of the present invention are prepared generally according to the method of Koehler and Milstein, Eur. J. Immunol. 6_, (1975) 292.
• EXAMPLE 1 A. Animal Immunisation
• Cell Fusion Spleen cells from the immune mice are harvested three days after boosting, by conventional techniques.
• the donor mouse selected is killed and surface-sterilised by immersion in 70% ethyl alcohol.
• the spleen is then removed and immersed in approximately 2.5 ml DMEM to which has been added 3% FCS.
• the spleen is then gently homogenised in a LUX homogenising tube until all cells have been released from the membrane, and the cells are washed in 5 ml 3% FCS-DMEM.
• the cellular debris is then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube.
• the debris is then rewashed in 5 ml 3% FCS-DMEM. 50 ml suspension are then made in 3% FCS-DMEM.
• the myeloma cell line used is NSO (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England.
• the myeloma cells are in the log growth phase, and rapidly dividing.
• Each cell line is washed using, as tissue culture medium, DMEM containing 3% FCS.
• the spleen cells are then spun down at the same time that a relevant volume of myeloma cells are spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet is then separately resuspended in 10 ml 3% FCS-DMEM.
• 0.1 ml of the suspension is diluted to 1 ml and a haemacytometer with phase microscope is used.
• 0.1 ml of the suspension is diluted to 1 ml with Methyl Violet-citric acid solution, and a haemacytometer and light microscope are used to count the. stained nuclei of the cells.
• 1 x 10 8 Spleen cells are then mixed with 5 x 107 myeloma cells, the mixture washed in serum-free DMEM hig in glucose, and centrifuged, and all the liquid removed.
• the resultant cell pellet is placed in a 37°C water-bath.
• the monoclonal antibodies from the clones are screened by the standard techniques for binding to the antigen, prepared as in the immunisation, and for specificity in a test battery of Herpes hominis Types I and II.
• the EIA immunoassay noted above may be used.
• DMEM-10% FCS was used to support growth in mid-log phase, to 1 litre volume. The culture was then allowed to overgrow, to allow maximum antibody production. The culture was then centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich supernatant collected.
• TRIS buffered supernatant was applied at a flow rate of 1 ml/min to a 1 ml column of Protein A-S.epharose, previously equilibrated with 0.1M TRIS buffer, pH 8.2. The column was then washed with 40 ml of 0.1M TRIS buffer.
• the monoclonal antibody was eluted with citrate buffer (0.1M sodium " citrate, pH 3.5) into sufficient- 1M TRIS buffer, pH 9.0, to raise the pH immediately to about 7.5.
• the eluate was dialysed in PBS, pH 7.4, at 4 C, and stored at -20 C.
• the monoclonal antibody specific against the antigen, prepared as above, is linked to an enzyme, viz. highly-purified alkaline phosphatase.
• alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25 M sodium phosphate buffer, pH 6.0, at +4 C. 18 mg p-benzoquinone were dissolved in 0.6 ml warm AR ethanol, and added to the dialysed. alkaline phosphatase. The benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. Unreacted benzoquinone and reaction by-products were then removed and the buffer exchanged by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column previously equilibrated in 0.15M sodiu chloride.
• Example i The general procedure of Example i was followed in each of 5 cases, with the following differences: Herpes simplex virus type 1 was used in the antigen in Examples 2 and 3, Herpes simplex virus type 2 in Examples 4 and 5, and Herpes simplex virus (common antigen) in Example 6. All these antigens were obtained from Cambridge University; their strain titles are HSVl, HSV2 and HSV . They were prepared by growth in baby hamster kidney cells; the preparation sequence in Example 6 involved harvesting, disruption etc.
• the animal immunisation step in Examples 2 and 3 comprised 10 5 pfu subcutaneously, 105 pfu ip after 3 weeks and 10 pfu iv after a further 4 weeks.
• Example 6 L ear and 10 pfu iv after 2 weeks.
• immunised mice were supplied.
• I Inn Example 4 1.25 x 10 spleen cells were used for fusion.
• Antibody production in Example 6 was conducted as follows:
• Balb/c mice were primed with pristane for at least 7 days, and were then injected with 10 cells of the monoclonal antibody-producing cell line. Ascitic fluid was harvested when the mice were swollen with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich ascites collected and stored at -20 C.
• Antibody purification in Example 6 was conducted as follows: Balb/c mice were primed with pristane for at least 7 days, and were then injected -with 10 cells of the monoclonal antibody-producing cell line. Ascitic fluid was harvested when the mice were swollen.with fluid but still alive. The fluid was centrifuged at 1200 g for approximately 10 minutes, the cells discarded and the antibody-rich ascites collected and stored at -20 C.
• Antibody conjugation in Examples 3 to 6 was conducted as follows: monoclonal antibody was dialysed with alkaline phosphatase (Sigma Type VII-T) , against 2 x 1000 ml of phosphate buffered saline (PBS), pH 7.4 at +4 C. After dialysis the volume was made up to 2.5 ml with PBS and 25 ⁇ l of a 20% glutaraldehyde in PBS solution added. The conjugation mixture was left at room temperature for 1.5 hours. After this time glutaraldehyde was removed by gel filtration on a Pharmacia PD-10 (Sephadex G-25M) column, previously equilibrated in PBS. The conjugate was eluted with 3.5 ml PBS.
• the conjugate was then dialysed vs 2 x 2000 ml of TRIS buffer (50 mM TRIS, 1 mM magnesium chloride, pH 8.0 + 0.02% sodium azide) at +4 C.
• TRIS buffer 50 mM TRIS, 1 mM magnesium chloride, pH 8.0 + 0.02% sodium azide
• To the dialysed conjugate was added 1/lOth its own volume of 10% BSA in TRIS buffer.
• the conjugate was then sterile filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial and stored at +4 C.
• the antibody of Example 2 was specific to HSV type 1 gD glycoprotein, of Example 3 to type 1 gC, of Examples 4 and 5 to 2gC, and of Example 6 to types 1 and 2.
• the antibodies were negative to other organisms, specifically E .
• Example 7 The general procedure of Example 1 may be followed to produce a monoclonal antibody broadly cross-reactive with an antigen of all types of the Herpes virus.
• Tests using the present invention are superior to existing tests, based on the following advantages: (i) greater accuracy; (ii) same day results, within an hour or two; (iii) reduction in amount of skilled labour required to administer laboratory procedures, resulting in reduced labour costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expenses; and (v) improved therapy based upon early, precise diagnosis.

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• 1984
  • 1984-10-19 GB GB848426467A patent/GB8426467D0/en active Pending
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