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
  • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
  • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• MONOCLONAL ANTIBODIES AND THEIR USE This invention relates to monoclonal antibodies and their use.
• Proteus vulgaris A third species, Proteus myxofaciens, has been isolated only from gypsy moth larvae.
• the extreme specificity of antigen-antibody reactions has made it possible to recognise differences between strains of a bacterial species, such as the above-mentioned species, that are indistinguishable on the basis of other phenotypic criteria.
• mirabilis accounts for the majority of Proteae infections in man, causing community-acquired, as well as hospital-acquired urinary tract infections.- Wound infections t pneumonia and septicemia can also occur.
• Proteus In addition to urinary tract infections, Proteus is known to cause gram-negative sepsis which is a bloodstream infection. It is one of the major infectious disease problems encountered in modern medical centres. While it can be transient and self-limited, severe gram-negative sepsis constitutes a medical emergency.
• the test for gram-negative sepsis involves processing blood and urine cultures and other procedures on occasion.
• blood culture tests are cumbersome. They require a day, and often several days, to return results. They require expert laboratory skills because of the complex nature of human blood which tends to interact non-specifically with many of the test reagents.
• urinary tract infections a microscopic examination is made, to determine the presence of micro-organisms as a preliminary screening. The microscopic examination cannot distinguish among the gram-negative bacteria.
• a second step is a urine culture to identify the organism isolated in the urine sample. A delay in diagnosis and initiation of treatment can result in serious complications.
• the present invention provides novel monoclonal antibodies for use in accurately and rapidly diagnosing samples for the presence of Proteus antigens and/or organisms.
• the present invention comprises monoclonal antibodies specific for an aritigen of Proteus? in particular, the antigens of Proteus mirabilis and the antigens of Proteus vulgaris, as well as a monoclonal antibody broadly cross-reactive with an antigen for each species of the genus Proteus.
• the invention also comprises labelled monoclonal antibodies for use in diagnosing the presence of the Proteus antigens, each comprising a monoclonal antibody -3-
• the label can be, for example, a radioactive isotope, enzyme, fluorescent compound, chemiluminescent compound, bioluminescent compound, ferromagnetic atom or particle.
• the invention further comprises the process for diagnosing the presence of Proteus antigens or organisms in a specimen, comprising contacting said specimen with the labelled monoclonal antibody in an appropriate immunoassay procedure.
• the invention is also directed to a therapeutic composition
• a therapeutic composition comprising a monoclonal antibody for an antigen of Proteus and a carrier or diluent, as well as kits containing at least one labelled monoclonal antibody to an antigen of a Proteus.
• the monoclonal antibodies of the present invention are prepared by fusing spleen cells from a mammal which has been immunised against the particular Proteus antigen, with an appropriate myeloma cell line, preferably NSO (uncloned) , P3NS1-Ag4/1, or Sp2/0 Agl4. The resultant product is then cultured in a standard HAT (hypoxanthine, a inopterin and thymidine) medium. Screening tests for the specific monoclonal antibodies are employed utilising immunoassay techniques which will be described below.
• the immunised spleen cells may be derived from any mammal, such as primates, humans, rodents (i.e. mice, rats and rabbits) , bovines, ovines and canines, but the * present invention will be described in connection with mice.
• the mouse is first immunised by injection of the particular Proteus antigen chosen, generally for a period of approximately eleven weeks. When the mouse shows sufficient antibody production against the antigen, as -4-
• the fusion can then be carried out utilising immunised spleen cells and an appropriate myeloma cell line.
• the fused cells yielding an antibody which gives a positive response to the presence of the particular Proteus antigen are removed and cloned utilising any of the standard methods.
• the monoclonal antibodies from the clones are then tested against standard antigens to determine their specificity for the particular Proteus antigen ⁇
• the monoclonal antibody selected, which is specific for the particular Proteus antigen or species, is then bound to an appropriate label. Amounts of antibody sufficient for labelling 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 labelled with various labels, as exemplified above. The present invention will be described with reference to the use of an enzyme-labelled monoclonal antibody.
• Examples of enzymes utilised as labels are alkaline phosphatase, glucose oxidase, galactosidase, peroxidase and urease.
• Such linkage with enzymes can be accomplished by any known method, such as the Staphylococcal Protein A method, the glutar ldehyde method, the benzoquinone method, or the periodate method.
• labelled monoclonal antibody is formed, testing is carried out employing one of a wide variety of conventional immunoassay methods. The particular method chosen will vary according to the monoclonal antibody and the label chosen. At the present time, enzyme immunoassays are preferred owing to their low cost, reagent stability, safety, sensitivity and ease of -5-
• EIA enzyme-linked im unosorbent assay
• Fluorescent-immunoassay is based on the labelling of antigen or antibody with fluorescent probes. A non-labelled antigen and a specific antibody are combined with identical fluorescently-labelled antigen. Both labelled and unlabelled antigen compete for antibody binding sites. The amount of labelled antigen bound to the antibody is dependent upon, and therefore a measurement of, the concentration of non-labelled antigen. Examples of this particular type of fluorescent-immunoassay include heterogeneous systems such as Enzyme-Linked Fluorescent Immunoassay, or homogeneous systems such as the Substrate-Labelled Fluorescent Immunoassay. The most suitable fluorescent probe r and the one most widely used, is fluorescein.
• fluorescein can be subject to considerable interference from scattering, sensitivity can be increased by the use of a fluorometer optimised for the probe utilised in the particular assay, and in which the effect of scattering can be minimised.
• fluorescence polarisation a labelled sample is excited with polarised light and the degree of polarisation of the emitted light is measured. As the antigen binds to the antibody, its rotation slows down and the degree of polarisation increases. Fluorescence polarisation is simple, quick and precise. However, at the present time, its sensitivity is limited to the micromole per litre range and upper nanomole per litre range with respect to antigens in biological samples.
• Luminescence is the emission of light by an atom or molecule as an electron is transferred to the ground state from a higher energy state.
• the free energy of a chemical reaction provides the energy required to produce an intermediate reaction or product in an electronically-excited state. Subsequent decay back to the ground.state is accompanied by emission of light.
• Bioluminescence is the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein or enzyme, such as luciferase, increases the efficiency of the luminescent reaction. The best known chemiluminescent substance is luminol.
• 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 Proteus 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 Proteus infection and they are used in amounts effective to cure; the amount may vary widely, depending 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 Proteus in various specimens. It is also possible to use the broadly cross-reactive monoclonal antibody which can identify the genus Proteus alone or as part of a kit containing antibodies that can identify other bacterial genera or species of Proteus and/or other bacteria. In the past, there have been difficulties in developing rapid kits because of undesirable cross-reactions of specimens; e.g. urine with antiserum. The use of monoclonal antibodies can eliminate these problems and provide highly specific and rapid tests for diagnosis.
• kits could be used in pathology laboratories for the rapid detection of gram-negative bacteria in urine, or on an out-patient basis.
• conjugated or labelled monoclonal antibodies for antigens and/or species of Proteus and other gram-negative bacteria can be utilised in a kit to identify such antigens and organisms in blood samples taken from patients for the diagnosis of possible Proteus or other gram-negative sepsis.
• the monoclonal test is an advance over existing procedures in that it is more accurate than existing tests; it gives "same day” results, provides convenience to the patient and improves therapy as a result of early, accurate diagnosis; and it reduces labour costs and laboratory time required for administration of the tests.
• the kit may be sold individually or included as a component in a comprehensive line of compatible immunoassay reagents sold to reference laboratories to detect the species and serotypes of Proteus.
• One preferred embodiment of the present invention is a diagnostic kit comprising at least one labelled monoclonal antibody against a particular Proteus antigen or species, as well as any appropriate stains, counterstains or reagents. Further embodiments include kits containing at least one control sample of a Proteus antigen and/or a cross-reactive labelled monoclonal antibody which would detect the presence of any of the given particular Proteus organisms in a particular sample.
• Monoclonal diagnostics which detect the presence of Proteus antigens can also be used in periodic testing of water sources, food supplies and food processing operations.
• the present invention describes the use of the labelled monoclonal antibodies to determine the presence of a standard antigen
• the invention can have many applications in diagnosing the presence of antigens by determining whether specimens, such as urine, blood, stool, water and milk, contain the particular Proteus antigen. More particularly, the invention could be utilised as a public health and safety diagnostic aid, whereby specimens such as water or food could be tested for possible contamination.
• API Analytical Profile Index (ref. Ayerst Laboratories)
• DMEM Dulbeccos Modified Eagles Medium
• FCS Foetal Calf Serum
• % T refers to vaccine concentrations measured in a 1 cm light path
• PBS Phosphate Buffered Saline
• Proteus mirabilis was obtained from the National Collection of Type Cultures (NCTC accession No. 60) and tested against standard reference typing sera to confirm -9-
• the antigen was removed from the lyophile, grown on blood agar, and tested by conventional biochemical (API) and agglutination tests with appropriate antisera to confirm it identity and purity. The cells were then transferred to DMEM, grown, and harvested for use as a source of antigen. The organisms were washed in formol saline by repeated centrifugation and were resuspended in formol saline.
• A. Animal Immunisation Six Balb/c mice were injected with the prepared antigen.
• mice were given 3 weekly intraperitoneal injections (0.05 ml of 80% T vaccine) of boiled killed Proteus mirabilis prepared as above, followed by 3 weekly intravenous injections and a further intravenous injection after a month.
• the mice were bled approximately six days after the last injection .and the serum tested for antibodies by assay.
• the conventional assay used for this serum titer testing was the enzyme-linked im unosorbent assay system.
• a positive titer of at least 10,000 a mouse was selected as a fusion donor and given a booster injection (0.02 ml of 80% T vaccine) intravenously, three days prior to splenectomy.
• the selected donor mouse was killed and surface sterilised by immersion in 70% ethyl alcohol.
• the spleen was then removed and immersed in approximately 2.5 ml of DMEM to which had been added 3% FCS.
• the spleen was then gently homogenised in a LUX homogenising tube until all cells had been released from the membrane and the cells were washed in 5 ml 3% FCS DMEM.
• the cellular debris was then allowed to settle and the spleen cell suspension placed in a 10 ml centrifuge tube. The debris was then rewashed in 5 ml 3% FCS DMEM. 50 ml of suspension were then made in 3% FCS DMEM.
• the myeloma cell line used was NSO (uncloned) , obtained from the MRC Laboratory of Molecular Biology in Cambridge, England. The myeloma cells were in the log growth phase, and rapidly dividing. Each cell line was washed using a tissue culture medium DMEM containing 3% FCS.
• the spleen cells were then spun down at the same time that a relevant volume of myeloma cells were spun down (room temperature for 7 minutes at 600 g) , and each resultant pellet was then separately resuspended in 10 ml 3% FCS DMEM.
• 0.1 ml of the suspension was diluted to 1 ml and a haemacytometer with phase microscope was used.
• 0.1 ml of the suspension was diluted to 1 ml with Methyl Violet-citric acid solution r and a haemacytometer and light microscope were used to count the stained nuclei of the cells.
• the resultant cell pellet was placed in a 37°C water bath. Over the period of one minute, 1 ml of a 50% w/v solution of polyethylene glycol 1500 (PEG) in saline
• each well contains 1.0 ml of the standard HAT medium (hypoxanthine, aminopterin, and thymidine) and a feeder layer of Balb/c
• the wells were kept undisturbed and cultured at 37°C in 9% C0 2 -air at approximately 100% humidity.
• the wells were analysed for growth utilising the conventional inverted microscope procedure, after about 5 to 10 days.
• a freshly-prepared stock solution of sterile 1.2% agar in double-distilled water with an equal volume of double-strength DMEM and additives was maintained at 45°C. This solution (10 ml) was then aliquoted into 10 cm Petri dishes, to form a base layer. An overlay of equal volumes of agar and cells in 18% FCS-DMEM was spread evenly over the base. The cells were allowed to multiply for approximately 10 days at 37°C, 7-9% CO TM , 9.5% RH. Viable separate colonies were picked off the agar surface and placed into 60 wells of a 96-well icrotitre tray in 18% FCS-DMEM. After a further period of growth, the supernatants were assayed for specific antibody by the standard enzyme immunosorbent assay.
• the monoclonal antibodies from the clones were screened by the standard techniques for binding to the antigen prepared as in the immunisation, and for specificity in a test battery of Proteus species and related genera bearing different antigens. Specifically, a grid of microtiter plates containing a representative selection of O-serotype organisms, i.e. Providencia, Shigella, Serratia and Salmonella, was prepared, boiled, and utilised as a template to define the specificity of the parent O-specific group. The EIA immunoassay noted above was used.
• F Antibody Production
• the tris buffered supernatant was applied at a flow rate of 1 ml/min to a 1 ml column of Protein A-Sepharose, 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. G.
• citrate buffer 0.1M sodium citrate, pH 3.5
• Enzyme-Monoclonal Linkage The monoclonal antibody specific against Proteus mirabilis antigen, prepared and screened as described above, is then bound to an appropriate enzyme; in this case, a highly purified alkaline phosphatase. This is accomplished by the benzoquinone conjugation method. 24 mg alkaline phosphatase (Sigma Type VII-T) were dialysed against 2 x 500 ml of 0.25M sodium phosphate buffer, pH 6.0, at +4°C. Para-benzoquinone, 18 mg, was dissolved in warm AR ethanol, 0.6 ml, and added to the dialysed alkaline phosphatase.
• the benzoquinone/alkaline phosphatase mixture was left in the dark at room temperature for 1 hour. After this time, unreacted benzoquinone and reaction by-products were removed and the buffer exchanged, by gel filtration on a Pharmacia ' PD-10 (Sephadex G-25M) column, previously equilibrated in 0.15M sodium chloride. The benzoquinone-activated alkaline phosphatase thus produced was sufficient for six 1.5 mg antibody conjugations. Monoclonal antibody was dialysed against 2 x 500 ml of 0.15M sodium chloride at +4"C.
• Dialysed antibody was added to 8 mg of benzoquinone-activated alkaline phosphatase, immediately followed by sufficient 1M sodium bicarbonate to give a final concentration of 0.1M.
• the conjugation mixture was left in the dark at +4°C for 48 hours. After this time, sufficient 1M lysine was added to give a final concentration of 0.1M.
• the conjugate was dialysed against 2 x 1000 ml of PBS + 0.02% sodium azide at +4°C. An equal volume of glycerol was added. The conjugate was sterile- filtered through a 0.22 ⁇ m membrane filter into a sterile amber vial and stored at +4°C. H. Monoclonal Antibody Conjugate Testing
• the enzyme immunoassay method was used for testing.
• This assay method comprises coating the wells of a standard polyvinyl chloride mi ⁇ rotiter tray with the antigen, followed by addition of monoclonal antibody enzyme conjugate, and finally addition of the enzyme substrate, para-nitrophenol phosphate.
• the monoclonal antibody was found to be specific for the antigen Proteus mirabilis.
• the monoclonal antibody was also tested and shown to be of the Class IgG3.
• the particular epitopic site to which the antibody attaches to the antigen can also be determined.
• the same enzyme immunoassay method can also be used to determine whether diagnostic specimens such as urine, blood, stool, water or milk contain the antigen.
• the antibody can first be bound to. the plate.
• Example 2 The same procedure as in Example 1 is utilized, in preparing the monoclonal antibody conjugate specific for the antigen of Proteus vulgaris, except for the following differences.
• Proteus vulgaris was obtained from the National Collection of Type Cultures (NCTC accession No. 4175) .
• the intraperitoneal injections were given weekly for 3 weeks, followed by intravenous injections for a further 3 weeks.
• the g cell fusion step 1 x 10 spleen cells were mixed with 6 10 myeloma cells.
• One agar method step was used in cloning.
• the sub-class was IgG2a.
• Example 3 The sub-class was IgG2a.
• Example 2 The same procedure as in Example 1 may be utilized in preparing a monoclonal antibody broadly cross-reactive with an antigen of many or all species of the genus Proteus, but using another Proteus obtained from the National Collection of Type Cultures.
• Tests using the present invention are superior to the 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 labor required to administer laboratory procedures, resulting in reduced labor costs; (iv) reduction in laboratory time and space used in connection with tests, resulting in reduced overhead expense; and (v) improved therapy based upon early, precise diagnosis.

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