WO1989009789A1 - Human monoclonal antibodies against rabies virus - Google Patents

Human monoclonal antibodies against rabies virus Download PDF

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Publication number
WO1989009789A1
WO1989009789A1 PCT/EP1989/000365 EP8900365W WO8909789A1 WO 1989009789 A1 WO1989009789 A1 WO 1989009789A1 EP 8900365 W EP8900365 W EP 8900365W WO 8909789 A1 WO8909789 A1 WO 8909789A1
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Prior art keywords
group
rabies
viruses
antibody
human
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PCT/EP1989/000365
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English (en)
French (fr)
Inventor
Gianluigi Devani
Fabrizio Marcucci
Elena Barbanti
Domenico Trizio
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Farmitalia Carlo Erba S.R.L.
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Priority claimed from GB888808153A external-priority patent/GB8808153D0/en
Priority claimed from GB888823081A external-priority patent/GB8823081D0/en
Application filed by Farmitalia Carlo Erba S.R.L. filed Critical Farmitalia Carlo Erba S.R.L.
Priority to KR1019890702268A priority Critical patent/KR900700135A/ko
Publication of WO1989009789A1 publication Critical patent/WO1989009789A1/en
Priority to FI895829A priority patent/FI895829A0/fi
Priority to DK616989A priority patent/DK616989D0/da

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    • 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
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/42—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum viral
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention concerns human monoclonal antibodies, hybrid cell lines that produce the antibodies and the use of the monoclonal antibodies.
  • Viruses of the rabies group of Rhabdoviridae comprise viruses that are infectious for nervous tissue of all warm-blooded animals. Rabies is usually contracted from the bite of an infected animal, but may also occur as a result of scratches or abrasions caused by an object contaminated with infected saliva, or rarely by penetration of mucous membranes, by exposure to a virus aerosol or the transplantation of infected tissues. See Harrison's Principles of Internal Medicine, 10th Edition, Petersdorf et al. , eds., McGraw-Hill Book Company, p. 1136 (1983).
  • the incidence of the disease in unvaccinated individuals bitten by rabid animals is about 15 percent and varies depending on the amount of the virus in the saliva and the location and depth of the wounds. After an incubation period, usually 30 to 70 days, rabies gives rise to an invariably fatal disease in man.
  • the first, or excitement, phase of the disease is characterised by fever, increasing agitation, spasmodic gross muscle contractions and generalised clonic or tonic convulsions with opisthotonos often precipitated by loud noises, bright lights or touch.
  • the most typical feature of the disease is severe, painful contractions of the pharyngeal muscles, initially precipitated by attempts to swallow fluids which leads to hydrophobia (fear of water). To avoid swallowing, patients allow saliva to drool from the mouth. Death usually follows a generalised convulsion with prolonged apnea.
  • peripheral white blood cell count is usually slightly elevated (12,000 to 17,000 per cubic centimeter) but may be normal or as high as 30,000 per cubic millimeter.
  • rabies As in any viral infection, the specific diagnosis of rabies depends upon (1) the isolation of virus from infected secretions [saliva, rarely cerebrospinal fluid (CSF), or tissue (brain)], (2) the serologic demonstration of acute infection, or (3) the demonstration of viral antigen in infected tissue, e.g., corneal impression smears, skin biopsies, or brain. Samples of brain obtained either on postmortem examination or from brain biopsy are subjected to (1) mouse inoculation studies for virus isolation, (2) fluorescent-antibody (FA) staining for viral antigen, and (3) histologic and/or electron microscopic examination for Negri bodies.
  • FA fluorescent-antibody
  • Another method for determination of rabies infection involves capturing the animal suspected of harboring the rabies virus, so that it may be killed and its brain examined by the FA technique. However, it is not always possible to find or capture the animal suspected of inflicting the bite. Thus, a need exists for a quick and accurate test for rabies infection which, in addition, does not require tissue biopsies.
  • rabies is an invariably fatal disease in man
  • prevention of rabies is of critical importance.
  • Control of rabies can be achieved through pre-exposure immunisation with a vaccine or through postexposure treatment.
  • Postexposure treatment rests mainly on three measures: (1) cleaning the wound with substances of proven lethal effect on rabies virus; (2) instillation or infiltration of antirabies serum around the wound; and (3) vaccine administration, including parenteral administration of antirabies serum.
  • Antirabies serum may be of heterologous or of human origin. With heterologous serum, occurrence of serum sickness is a frequent event (15-45% in persons given heterologous serum of equine origin) and requires administration of an anti-histamine drug.
  • the use of antirabies serum of human origin is increasing because of the lower incidence of harmful side effects. However, its supply is limited by the fact that anti ⁇ erum collection is from human volunteers. Thus, it would represent a considerable advance to produce jLn vitro, in unlimited amounts, human anti-rabies antibodies of reproducible specificity and affinity. Somatic cell hybridisation is the method of choice to achieve this goal. Thus, in one aspect, this invention is directed toward the production of such human anti-rabies antibodies.
  • Procedure (I) suffers from the disadvantage that EBV-transformed cell lines are extremely unstable in antibody production and, moreover, are very difficult to clone.
  • Procedure (II) suffers from the disadvantage that mouse/human hybridomas tend to segregate preferentially human chromosomes and, thus, are highly unstable in sustaining human antibody production.
  • the present invention provides human monoclonal antibodies which:
  • the invention further provides hybridoma cell lines and progeny thereof which produce the human monoclonal antibodies of the present invention.
  • a hybridoma cell line is prepared by a process comprising fusing immortalizing cells with cells derived from a human which produce antibody which binds specifically to viruses of the serotype 1 group of the rabies group of Rhabdoviridae.
  • T e invention additionally provides a process for the preparation of a human monoclonal antibody of the invention, which process comprises culturing a hybridoma cell line or progeny thereof according to the invention and recovering the monoclonal antibody thus-produced.
  • the invention also relates to pharmaceutical compositions comprising a human monoclonal antibody of the invention and a pharmaceutically acceptable carrier or diluent; and to a method of detecting whether a sample contains a virus of the serotype 1 group of the rabies group of Rhabdoviridae, which method comprises contacting the sample with a monoclonal antibody of the invention and detecting whether an antibody-antigen complex is formed.
  • the antibodies may be of any immunoglobulin class, preferentially IgG or IgM.
  • Monoclonal antibody can be obtained which recognises an epitope common to at least three laboratory strains of rabies virus - Pitman-Moore, CVS and ERA - and which is able to at least partially protect cells from infection in conditions where the virus •has already bound to the cell-surface.
  • Figure 1 shows the detection of neutralizing anti-rabies virus antibodies in hybridoma supernatants.
  • A, B, C and D represent negative controls—A: fresh complete RPMI medium; B: spent medium from FD5 cells; C: spent medium from a murine fibroblast cell line; D: supernatant from a hybridoma producing human monoclonal anti-tetanus-toxoid IgM antibodies—and E and F represent positive controls—E: serum from the PBL donor (tested at a 1:10 final dilution); F: commercially available anti-rabies antiserum (tested at a 1:100 final dilution). Hybridoma supernatants were tested at a 1:10 final dilution.
  • Figure 2 shows the detection of neutralizing anti-rabies virus antibodies in supernatants of clones derived from hybridoma 208.
  • a to F represent controls as above.
  • Supernatants of the clones were tested at a 1:10 final dilution. Three panels were tested: a: neutralization was evaluated upon incubation of supernatants with 10 infectious rabies virus particles b: with 10 3 infectious rabies virus particles; and c: with 10 2 infectious rabies virus particles.
  • Figure 3 shows immunofluorescence studies with antibodies produced by clone 208/97 (1, 3) or with a polyclonal anti-nucleocapside antiserum (2, 4) on normal cells (3, 4) or cells infected with rabies virus (1, 2). Immunofluorescence tests were carried out on fresh (A) or fixed (B) cells.
  • Figure 4 shows the neutralizing potency of the monoclonal anti-rabies antibodies produced by clone 208/97 and of a reference equine anti-rabies serum.
  • ⁇ antibodies produced by clone 208/97 (10 g/ml final concentration);
  • Figure 5 shows the antiviral activity of the monoclonal antibodies produced by clone 208/97 and of a reference antiserum on rabies virus particles adsorbed to target cells.
  • B monoclonal antibodies produced by clone 208/97;
  • Q reference equine anti-rabies serum.
  • rabies virus means viruses of the serotype 1 rabies group of Rhabdoviridae as defined in the WHO Technical Report Series, No. 709 (1984), and comprises both “street rabies viruses”, as used to designate the agents of the naturally occurring disease, as well as “fixed rabies viruses”, as used to designate rapidly multiplying strains used in vaccine production which have lost their infectivity for salivary gland tissue after passage in the laboratory.
  • the terms “permanent” and “stable” mean viable over a prolonged time, typically at least about six months.
  • the invention enables stable, permanent hybridoma cell lines to be provided which maintain the ability to produce the specified monoclonal antibody through at least 25 passages.
  • the term "monoclonal antibody” refers to an antibody selected from antibodies whose population is substantially homogeneous, i.e. the individuals of the antibody population are identical except for naturally occurring mutations.
  • antibody is also meant to include intact molecules as well as fragments thereof, such as Fab and F(ab') 2 , which are capable of binding antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of antibody, clear more rapidly from the circulation and may have less non ⁇ specific tissue binding than intact antibody. It will be appreciated that Fab, F(ab') 2 and other fragments of the monoclonal antibody of the present invention may be used as well as the intact antibody for the detection and treatment of rabies viral infection according to the methods of the present invention.
  • neutralise is used to denote the ability of antibody-containing supernatants to block the capacity of infectious rabies virus to infect target cells.
  • Such a monoclonal antibody is secreted by a hybridoma cell line which has been prepared using cells of an immortalizing cell line and cells derived from a human which produce antibody having the specificity (a) above.
  • the immortalizing cell line is a cell line which, for practical purposes, can be maintained perpetually in cell culture. In other words, it is stable and permanent and, when fused with cells which do not exhibit these properties, is able to confer the properties on the fusion product.
  • any appropriate immortalizing cell line may be used.
  • a plasmacytoma (myeloma) or lymphoblastoid cell line of mammalian origin may be employed such as a lymphoblastoid cell line of human origin.
  • a preferred type of cell line is a human hypoxanthine-phosphoribosyl-transferase (HPRT) deficient and ouabain-resistant lymphoblastoid cell line.
  • HPRT human hypoxanthine-phosphoribosyl-transferase
  • One such cell line is particularly preferred. This is the cell line FD5 which has been deposited at the European Collection of Animal Cell Cultures (ECACC), Porton Down, GB on 19 February 1988 under accession number 87061701.
  • This HPRT-deficient and ouabain-resistant mutant cell line was derived from the human B-lymphoblastoid cell line GM 1500A (obtained from the Human Cell Depository,
  • HPRT-deficient mutant was obtained through selection in the presence of 20 g/ml 8-azaguanin of an ethyl-methane sulphonate mutagenized cell population.
  • the HPRT-deficient line was cultured in medium containing increasing concentrations of ouabain (up to 10 _5 M).
  • FD5 cells produce their own low amounts of IgG antibodies ( ⁇ 75 ng/10 6 cells/day). They grow in suspension as a typical lymphoblastoid line, forming large cell clumps.
  • the growth medium consists of RPMI 1640
  • the immortalizing cells are fused with cells derived from a human which produce antibody which binds specifically to viruses of the serotype 1 group of the rabies group Rhabdoviridae.
  • These antibody-producing cells are lymphocytes which may or may not have been transformed with a virus.
  • Epstein-Barr virus (EBV) is used for transformation.
  • EBV Epstein-Barr virus
  • B-lymphocytes which are separated and fused with the immortalizing cells.
  • the antibody-producing cells are generally obtained from a human volunteer immunised with respect to a rabies virus of serotype group 1. A commercially available rabies virus vaccine may be used for this purpose.
  • Peripheral blood lymphocytes (PBL) are obtained from the volunteer.
  • the B-lymphocytes or at least a B-cell rich fraction of the PBL is generally obtained and transformed with a virus if desired.
  • the lymphocytes may be restimulated jj vitro with a rabies virus of the serotype 1 group such as an active or inactive (vaccine) rabies virus, in the absence or presence-of a mitogenic substance-, before hybridisation or, if appropriate, transformation.
  • the fusion products are screened for those secreting the desired monoclonal antibody.
  • preferred strategy not intended to limit the invention in any manner, for preparing and identifying hybrids that produce antibodies of the invention is as follows.
  • rabi ' es virus vaccine (Rasilvax (Trade Mark), Sclavo, Italy) consisting of an inactivated and lyophilized suspension of rabies virus (strain Pitman-Moore/Wistar PM/W 381503-3M) .
  • PBL obtained from this donor are separated into a B-cell enriched and a B-cell depleted fraction through panning on goat anti-human immuneglobulin- coated plastic dishes.
  • the cells of the adherent,- B-cell enriched fraction are transformed with EBV and cultured at 1000 cells per microtiter well using irradiated (3000 Rad) mouse peritoneal cells as feeder cells.
  • Supernatants from the resulting lymphoblastoid cell lines are screened after approximately 15 days in an ELISA for immunoglobulin production and then in an ELISA against Rasilvax for the detection of antibodies binding to determinants defined by the vaccine.
  • Supernatants that score positive in both assays and which exhibit the highest activities in the latter assay are pooled, expanded, and finally fused to HPRT-deficient and ouabain- resistant FD5 cells.
  • Hybrids are selected in a tissue culture medium containing aminopterin and ouabain.
  • Supernatants are screened in a Rasilvax-ELISA as described in the Example below. Those exhibiting the highest activities in this assay are treated for their ability to neutralise infectious rabies virus.
  • Cells from wells producing the desired antibodies are cloned by limiting dilution in wells containing irradiated mouse peritoneal cells as feeder cells.
  • the progeny of clones producing the desired antibodies are grown i_n vitro in suitable culture media in tissue culture flasks or in a hollow fiber tissue culture device (e.g. Acusyst-Jr., Endotronics, Coon Rapids, Minnesota) or in vivo in immunodeficient laboratory animals.
  • the antibody may be separated from the culture medium or body fluid, as the case may be, by techniques such as ammonium sulfate precipitation, ion exchange chromotography, affinity chromatography, high- performance liquid chromatography or by other techniques known to those of ordinary skill in the art.
  • the preferred strategy therefore, it is possible to produce human monoclonal antibodies which are characterised in that: a) their population is substantially homogeneous; b) they are produced by immortal cells which are themselves hybrids between an immortal cell line and an antibody-producing human cell; c) they bind to determinants defined by a rabies virus vaccine; and d) they neutralise infectious rabies virus so that it can no longer infect target cells.
  • the monoclonal antibodies of the invention find therapeutic application as part of a suitable post-exposure protocol in individuals at risk to develop rabies. They can be instilled or infiltrated around the wound or administered parenterally together with a vaccine.
  • the monoclonal antibodies of this invention may be formulated in pharmaceutical compositions by including appropriate amounts of the monoclonal antibody together with a pharmaceutically acceptable carrier or diluent.
  • these carriers and diluents include or alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, 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 can also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. See, generally. Remington's Pharmaceutical Sciences, 16th Ed., Mack, eds., 1980.
  • the monoclonal antibody is preferentially by methods presently employed with rabies immune globulin of either human or heterologous origin.
  • the monoclonal antibody may be instilled or infiltrated around the wound of individuals bitten by aminals and at risk of developing rabies. Moreover it may be administered intramuscularly in a single dose. These treatments can be followed by a complete course of vaccine, see WHO Technical Report Series.
  • the monoclonal antibody is typically used at doses equivalent to those currently employed with rabies immune globulin of human origin, see WHO Technical Report Series.
  • Monoclonal antibody of the invention may therefore be administered at doses of 0.4-1.6 mg/kg body weight which corresponds to 20-40 International Units (I.u.) of rabies immune globulin of human origin.
  • the monoclonal antibodies of the present invention are particularly suited for use in i munoassays wherein they may be utilized in liquid phase or bound to a solid phase carrier.
  • the monoclonal antibody in these immunoassays can be detectably labeled in various ways.
  • carrier to which the monoclonal antibody of the present invention can be bound and which can be used in detecting the presence of the rabies virus.
  • Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agoroses and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • suitable carriers for binding monoclonal antibody or will be able to ascertain the same by the use of routine experimentation.
  • labels and methods of labeling known to those of ordinary skill in the art.
  • Examples of the types of labels which can be used in the present invention include, but are not limited to, enzymes, radioisotopes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds and metal chelates.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the monoclonal antibody, or will be able to ascertain the same by the use or routine experimentation.
  • the binding of these labels to the monoclonal antibody can be accomplished using standard techniques commonly known to those of ordinary skill in the art.
  • One of the ways in which a monoclonal antibody of the present invention can be detectably labeled is by linking the same " to an enzyme.
  • enzymes which can be used to detectably label the monoclonal antibodies of the present invention include malate dehydrogenase, staphyloccocal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alp a-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholine esterase.
  • the monoclonal antibody of the present invention can also be labeled with a radioactive isotope which can then be determined by such means as the use of a gamma counter or a scintillation counter.
  • a radioactive isotope which are particularly useful for the purpose of the present invention are: 3 H, 125 I, 131 I, 32 P, 35 S, 14 C, 51 Cr, 36 C1, 57 Co, 58 Co, 59 Fe, and 7S Se.
  • the monoclonal antibody it is also possible to label the monoclonal antibody with a fluorescent compound.
  • a fluorescent compound When the fluorescently labeled monoclonal antibody is exposed to light of the proper wave length, its presence can then be detected due to the fluorescence of the dye.
  • fluorescent labelling compounds are fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, ophthaldehyde and fluorescamine.
  • the monoclonal antibody of the invention can also be detectably labeled using fluorescent emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the antibody molecule using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the monoclonal antibody of the present invention also can be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged monoclonal antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, i idazole, acridinium salt and oxalate ester.
  • a bioluminescent compound may be used to label the monoclonal antibody of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent monoclonal antibody is determined by detecting the presence of luminescence.
  • Important bioluminescent compounds for purposes of labeling are luciferin, luciferase and aequorin.
  • Another technique which may also result in greater sensitivity when used in conjunction with the present invention consists of coupling the monoclonal antibody of the present invention to low molecular weight haptens.
  • the haptens can then be specifically detected by means of a second reaction.
  • the rabies virus which is detected by the monoclonal antibody of the invention may be present in biological fluids or tissues.
  • Any sample, obtained from an infected animal or human, containing the detectable yet unknown amount of the virus can be used.
  • the sample is a liquid, such as, for example, the saliva of an infected animal, cerebrospinal fluid, blood, serum, urine and the like, or a solid or semi-solid, such as, for example, tissue, feces and the like.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement therewith one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay to be used.
  • a similar kit may be prepared comprising compartmentalized carrier means having one or more container means comprising separate elements suitable for therapeutic use according to the present invention.
  • immunoassays which can be used or incorporated in kit form are many. Typical examples of some of the immunoassays which can utilize the antibodies of the invention are competitive assays and immunometric, or sandwich, immunoassays.
  • immunometric assay or "sandwich immunoassay” it is meant to include simultaneous sandwich, forward sandwich and reverse sandwich immunoassays. These terms are well understood by those of ordinary skill in the art. Those of ordinary skill in the art will also appreciate that the monoclonal antibody of the present invention may be useful in other variations and forms of immunoassays which are presently known or which may be developed in the future. These are intended to be included within the scope of the present invention.
  • a sample is first incubated with a solid phase immunoabsorbent containing monoclonal antibody(ies) against the rabies antigen. Incubation is continued for a period of time sufficient to allow the antigen in the sample to bind to the immobilized antibody in the solid phase. After the first incubation, the solid phase immunoabsorbent is separated from the incubation mixture and washed to remove excess antigen and other interfering substances, such as non-specific binding proteins, which also may be present in the sample. Solid phase immunoabsorbent containing rabies antigen bound to the immobilized antibody is subsequently incubated for a second time with soluble labelled antibody or antibodies.
  • the process comprises, in more detail:
  • step (b) adding to the mixture after said incubation of step (a) the detectably labelled antibody or antibodies and incubating the new resulting mixture for a time and under conditions sufficient to allow the labelled antibody to bind to the solid phase immunoabsorbent;
  • step (c) separating the solid phase immunoabsorbent from the mixture after the incubation in step (b) ;
  • a reverse sandwich assay the sample is initially incubated with labelled antibody(ies) , after which the solid phase immunoabsorbent containing multiple immobilized antibodies is added thereto, and a second incubation is carried out.
  • the initial washing step of a forward sandwich assay is not required, although a wash is performed after the second incubation.
  • Reverse sandwich assays have been described, for example, in United States Patents 4,098,876 and 4,376,110.
  • the process comprises, in more detail:
  • step (b) adding to the mixture after the incubation of step (a) the solid phase bound antibodies and incubating the new resulting mixture for a time and under conditions sufficient to allow antigen bound to the labelled antibody to bind to the solid phase antibodies;
  • step (c) separating the solid phase immunoabsorbent from the incubating mixture after the incubation in step (b); and (d) detecting either the labelled antibody bound to the solid phase immunoabsorbent or detecting the labelled antibody not associated therewith.
  • a simultaneous sandwich assay the sample, the immunoabsorbent having multiple immobilized antibodies thereon and labelled soluble antibody or antibodies are incubated simultaneously in one incubation step.
  • the simultaneous assay requires only a single incubation and has a lack of washing steps.
  • the use of a simultaneous assay is by far the preferred method. This type of assay brings about ease of handling, homogeneity, reproducibility, linearity of the assays and high precision.
  • the sample containing antigen, solid phase immunoabsorbent with immobilized antibodies and labelled soluble antibody or antibodies is incubated under conditions and for a period of time sufficient to allow antigen to bind to the immobilized antibodies and to the soluble antibody(ies) .
  • Typical conditions of time and temperature are two hours at 45°C, or twelve hours at 37°C.
  • Antigen typically binds to labelled antibody more rapidly than to immobilized antibody, since the former is in solution whereas the latter is bound to the solid phase support. Because of this, labelled antibody may be employed in a lower concentration than immobilized antibody, and it is also preferable to employ a high specific activity for the labelled antibody. For example, labelled antibody might be employed at -a concentration of about 1-50 ng/per assay, whereas immobilized antibody might have a concentration of 10-500 ng/per assay per antibody. Where radiolabelled, the antibody might have a specific activity with, for instance, one radioiodine per molecule, or as high as two or more radioiodines per molecule of antibody.
  • the process comprises, in more detail:
  • step (a) simultaneously forming a mixture comprising the sample, together with the solid phase bound antibody and the soluble labelled antibody or antibodies;
  • step (b) ' incubating the mixture formed in step (a) for a time and under conditions sufficient to allow antigen in the sample to bind to both immobilized and labelled antibodies;
  • step (c) separating the solid phase immunoabsorbent from the incubation mixture after the incubation; and .
  • concentrations of labelled and immobilized antibodies, the temperature and time of incubation as well as other assay conditions can be varied, depending on various factors including the concentration of antigen in the sample, the nature of the sample, and the like. Those skilled in the art will be a ⁇ ?le to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • the solid phase immunoabsorbent is removed from the incubation mixture.
  • This can be accomplished by any of the .known separation techniques, such as sedimentation and centrifugation.
  • Detection can be performed by a scintillation counter, for example, if the label is a radioactive gamma-emitter, or by a fluorometer, for example, if the label is a fluorescent material.
  • the detection can be accomplished by colorimetric methods which employ a substrate for the enzyme.
  • solid phase immunoabsorbents There are many solid phase immunoabsorbents which have been employed and which can be used in the present invention.
  • Well known immunoabsorbents include beads formed from glass, polystyrene, polypropylene, dextran, nylon and other materials; tubes formed from or coated with such materials and the like.
  • the immobilized antibodies can be either covalently or physically bound to the solid phase immunoabsorbent, by techniques such as covalent bonding via an amide or ester linkages, or by absorption.
  • suitable solid phase immunoabsorbents and methods for immobilizing antibodies thereon or will be able to ascertain such, using no more than routine experimentation.
  • a vaccinated human volunteer known to be a high responder for antibody production against rabies virus was booster-immunized with Rasilvax. Fifty ml of venous blood were drawn from the cubital vein after four days.
  • the heparinized blood was diluted 1:1 with Ca ++ -Mg ++ free Earle's Balanced Salt Solution (EBSS) and then gradient centrifuged for 40 minutes at 300 x g using commercially available Ficoll/Hypaque (d - 1.077, Lymphoprep, Immuno, Pisa, Italy).
  • EBSS Ca ++ -Mg ++ free Earle's Balanced Salt Solution
  • Buffy coat lymphocytes were suspended (3 x 10 6 cells/ml) in complete RPMI medium (RPMI 1640 (Flow, McLean, Virginia) supplemented with 20% fetal calf serum (Flow, heat inactivated at 56°C for 30 minutes), 1 mM MEM sodium pyruvate (Gibco, Paisley, Scotland), MEM nonessential amino acids (Gibco), 100 ug/ml of penicillin, 100 ug/ml of streptomycin, 1.5 ug/ml of amphotericin B and additional gluta ine (2mM)) and incubated (5 ml/dish) at 4°C for 70 min in a plastic Petri dish (100 mm plastic dishes, Falcon 1005, Becton Dickinson, Mountain View, California) that had been previously coated with goat antihuman immunoglobulin antiserum (Cappel, Malvern, Pennsylvania, 20 ug/ml in 20 mM phosphate buffer, 5 ml/dish).
  • EBV-transformed cell cultures that had been obtained as described in the preceding section and cells from the HPRT-deficient and ouabain-resistant FD5 cell line. This latter cell line secretes negligible amounts of IgG.
  • the fusion mixture contained polyethylene glycol (PEG) 1500 (Serva, Heidelberg, West Germany) 40% w/v in complete RPMI medium without fetal calf serum.
  • Parent cells were washed twice in serum-free complete RPMI medium at room temperature and subsequently resuspended and combined at a ratio of 1:1 of EBV-transformed cells: FD5 cells in serum-free RPMI warmed at 37°C in a conical 50 ml centrifuge tube (Falcon No. 2070).
  • the mixture was diluted slowly with serum-free complete RPMI medium. The volume was adjusted to give a cell density of 5 x 10 5 FD5 cells/ml. 200 ul of this suspension was seeded into each well of 96-well plates that had been previously seeded with irradiated mouse peritoneal cells (5 x 10 4 cells/well). Plates containing the fusion products were then incubated for 24 hours at 37°C, 5% C0 2 .
  • the culture medium was then replaced with complete RPMI medium containing hypoxanthine (10 ⁇ 4 M), aminopterin (4 x 10 ⁇ 7 M), thymidine (1.6 x 10 "5 M) and ouabain (10 "6 M).
  • This medium is hereafter referred to as HAT-0 medium.
  • HAT-0 medium In this culture medium, only hybridomas can survive, since FD5 cells are killed in the presence of aminopterin and EBV-transformed cells are killed in the presence of ouabain.
  • the plates were then further incubated at 37°C, 5% CO for 5-6 weeks. During this period, the cultures were fed every three days with fresh medium. Growing hybrids were visible by day 15.
  • infectious virus particles were allowed to adsorb for 1 hour on to target chicken-embryo related (CER) cells under conditions (4°C) that precluded virus internaliziation.
  • CER chicken-embryo related
  • cells were washed free of unadsorbed virus and fresh culture medium with or without monoclonal antibody 208/97 or polyclonal antiserum was added to the cells.
  • PBS Phosphate-buffered saline
  • BSA Armour Pharmaceutical Co., Kanakee, Illinois
  • Rasilvax-ELISA Flat bottom microtiter plates were coated at 100 ul/well with 50 ug/ml of Rasilvax diluted in 50 mM bicarbonate buffer, pH 9.6. After an overnight incubation at 4°C, the plates were washed twice with WB. PBS with 1% BSA was then added to each well to saturate unoccupied plastic sites and the plates further incubated for 30 minutes at room temperature. Thereafter, plates were washed as above. Test supernatants (100 ul) were added to each well, and the plates incubated for 60 minutes at 37°C, then washed as above.
  • Peroxidase substrate addition and color development was as for Ig-ELISA.
  • CER cells were grown in Eagle's Minimum Essential Medium (EMEM) supplemented with 5% newborn calf serum (NCS) in eight chamber microtissue chamber/slides (Miles) for 24 h at 37°C, 5% C0 2 . Cells were then washed 3 times with serum-free EMEM and then infected with CVS (challenge virus strain) fixed rabies virus diluted in EMEM 1% NCS for 1 h at 37°C.
  • EMEM Eagle's Minimum Essential Medium
  • NCS newborn calf serum
  • Virus-neutralizing antibodies were measured by a direct immunofluorescene technique. (RFFIT) .
  • the neutralizing effect of hybridoma supernatants was determined by incubating 200 l of the indicated dilution of hybridoma or control supernatant with an equal volume of EMEM 1% FCS containing different numbers of infectious rabies virus particles for 1 hour at 37°C.
  • Rabies virus was CVS or ERA strain of SAD virus propagated in BHK-21 cells. At the end of the incubation period each supernatant mixture was added to CER cells grown in microtissue chamber/slides. After lh at 37°C, the inocula were removed and the monolayers were washed with EMEM. Cells were further incubated at 37°C, 5% C0 2 for a time period sufficient to have 100% of the cells in control cultures (virus alone) infected. The percentage of infected cells was then determined in direct IF on fixed cells using FITC-conjugated rabbit anti-rabies virus nucleocapsid IgG as described in section 3.
  • Results are expressed as per cent fluorescence inhibition in cell cultures treated with antibody/virus mixtures versus cells treated with virus alone.
  • 10 infectious CVS rabies virus particles were allowed to adsorb to CER cells grown in microtissue chamber/slides by incubation for 1 h at 4°C. Cells were then washed with serum-free EMEM and in-cubated for 1 h at 37°C with either prewarmed EMEM 1% FCS alone or EMEM 1% FCS containing monoclonal antibodies 208/97 or an international reference anti rabies serum. Cells were then washed as above and further incubated for 72 h at 37°C with EMEM 5% NCS. The number of infected cells was then determined as previously described. 5. Chromosome preparation
  • Chromosome preparations of the 208/97 hybridoma cells were made by use of the air drying technique described by Ishihara et al., Cancer Res., 22:375-379, (1962).
  • the optical density (O.D.) background (determined in the absence of the first antibody) ranged typically between 0.2 and 0.3.

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PCT/EP1989/000365 1988-04-07 1989-04-05 Human monoclonal antibodies against rabies virus WO1989009789A1 (en)

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KR1019890702268A KR900700135A (ko) 1988-04-07 1989-04-05 광견병 비루스 세포를 특이적으로 인지하는 사람 모노클로날 항체, 이의 생성세포, 그 항체를 함유하는 제제 및 이들의 제조방법
FI895829A FI895829A0 (fi) 1988-04-07 1989-12-05 Maenskliga monokloniska antikroppar mot rabiesvirus.
DK616989A DK616989D0 (da) 1988-04-07 1989-12-07 Humane monoklonale antistoffer, der specifikt genkender rabiesvirusceller, celler, der prducerer denne virus, formuleringer, der indeholder virusen, samt fremstilling af disse antistoffer

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GB888808153A GB8808153D0 (en) 1988-04-07 1988-04-07 Human monoclonal antibodies specifically recognizing rabies virus cells cells producing same formulations containing same & production of all thereof
GB8823081.8 1988-09-30
GB888823081A GB8823081D0 (en) 1988-09-30 1988-09-30 Human monoclonal antibodies specifically recognizing rabies virus cells cells producing the same formulations containing the same

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0445625A1 (de) * 1990-03-03 1991-09-11 BEHRINGWERKE Aktiengesellschaft Humane monoklonale Antikörper gegen Tollwutviren, ihre Herstellung und Verwendung
WO2001088132A2 (en) * 2000-05-16 2001-11-22 Thomas Jefferson University Rabies virus-specific neutralizing human monoclonal antibodies and nucleic acids and related methods
US7071319B2 (en) 2000-05-16 2006-07-04 Thomas Jefferson University Recombinant antibodies, and compositions and methods for making and using the same

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2086937A (en) * 1980-11-07 1982-05-19 Wilstar Inst Production of Human Monoclonal Antibodies by Human Hybridomas
CA1212913A (en) * 1981-06-29 1986-10-21 Queen's University At Kingston Human monoclonal antibodies
WO1987000179A1 (en) * 1985-07-03 1987-01-15 The Salk Institute For Biological Studies Synthetic peptide-based anti-rabies compositions and methods

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2086937A (en) * 1980-11-07 1982-05-19 Wilstar Inst Production of Human Monoclonal Antibodies by Human Hybridomas
CA1212913A (en) * 1981-06-29 1986-10-21 Queen's University At Kingston Human monoclonal antibodies
WO1987000179A1 (en) * 1985-07-03 1987-01-15 The Salk Institute For Biological Studies Synthetic peptide-based anti-rabies compositions and methods

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, vol. 80, 1985, abstract no. 77161, F.C.M. Van Meel et al.: "Human and chimpanzee monoclonal antibodies", & J. Immunol. Methods 80(2): 267-276, 1985 *
Chemical Abstracts, vol. 107, 1987, (Columbus, Ohio, US), B. Dietzschold et al.: "Mechanisms of rabies virus neutralization by glyco-protein-specific monoclonal antibodies", page 600 *
The Journal of Immunology,vol. 133, no. 4, October 1984, The American Association of Immunologists (US), D. Emanuel et al.: "A human monoclonal antibody to cytomegalovirus (CMV)", pages 2202-2205 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0445625A1 (de) * 1990-03-03 1991-09-11 BEHRINGWERKE Aktiengesellschaft Humane monoklonale Antikörper gegen Tollwutviren, ihre Herstellung und Verwendung
WO2001088132A2 (en) * 2000-05-16 2001-11-22 Thomas Jefferson University Rabies virus-specific neutralizing human monoclonal antibodies and nucleic acids and related methods
WO2001088132A3 (en) * 2000-05-16 2002-07-18 Univ Jefferson Rabies virus-specific neutralizing human monoclonal antibodies and nucleic acids and related methods
US6890532B2 (en) 2000-05-16 2005-05-10 Thomas Jefferson University Rabies virus-specific neutralizing human monoclonal antibodies and nucleic acids and related methods
US7071319B2 (en) 2000-05-16 2006-07-04 Thomas Jefferson University Recombinant antibodies, and compositions and methods for making and using the same

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AU3432389A (en) 1989-11-03
KR900700135A (ko) 1990-08-11
ES2013665A6 (es) 1990-05-16
FI895829A0 (fi) 1989-12-05
IL89857A0 (en) 1989-12-15
EP0404836A1 (en) 1991-01-02
GR890100219A (el) 1990-01-31
PT90218A (pt) 1989-11-10

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