AU634030B2 - Human monoclonal antibodies against rabies viruses, the production and the use thereof - Google Patents

Description

Form COMMONWEALTH O- AU8 PATENTS ACT 1952b /I COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Ludged.

Accepted: Published: Priority Related Art Name of Applicant Address of Applicant Actual Inventor Address for Sevice BEHRINGWERKE AKTIENGESELLSCHAFT D-3550 Marburg, Federal Republic of Germany KARLHEINZ ENBLE, JOACHIM HILFENHAUS, ROLAND KOHLER, ROLAND KURRLE and FRIEDRICH-ROBERT SEILER.

WATERMARK PATENT TRADEMARK ATTORNEYS.

LOCKED BAG NO. 5, HAWTHORN, VICTORIA 3122, AUSTRALIA Complete Specification for the invention entitled: HUMAN MONOCLONAL ANTIBODIES AGAINST RABIES VIRUSES, THE PRODUCTION AND THE USE THEREOF.

The following sta ament is a full description of this invention, including the best method of performing it known to la BEHRINGWERKE AKTIENGESELLSCHAFT HOE 90/B 010 Ma 809 Dr. Lp/rd Description Human monoclonal antibodies against rabies viruses, the production and the use thereof The present patent application relates to human monoclonal antibodies against rabies viruses, and the use thereof in diagnosis, prevention and therapy. Antibodies against the epitope, defined by the human monoclonal antibody TW-1, on the 67 kD glycoprotein of the virus envelope give a better protection against the outbreak of rabies, after infection has taken place, than commercial preparations of human immunoglobulins.

Rabies is an encephalomyelitis having a lethal course, which is caused by rabies virus, a member of the Rhabdoviridae family. Especially in less developed countries, the disease is a big problem and, for example, in India more than 20,000 deaths per year are ascribed to rabies according to cautious estimates. The infection often takes place via a bite by infected animals (Canidae, Chiroptera). The first target cell for the virus seems to be muscle cells at the place of entry.

After entry into peripheral nerves, the virus migrates from the infection site by retrograde axoplasmatic transport to the central nervous system and the spinal cord. After viral replication in the central nervous system, the virus migrates via the nerve paths to various target tissues in the body, e.g. salivary glands.

Owing to the long incubation time, in relation to other viral infections, of about 30 to 90 days until rabiesspecific symptoms appear, post-exposure vaccination is possible. In order to achieve fast protection until the active immunization is effective, a passive immunization 2 with homologous or heterologous serum preparations together with the active immunization is urgently recommended by the WHO. In underdeveloped countries, especially preparations from horse sera are used, anaphylactic reactions being a serious problem.

In contrast to polyclonal preparations, monoclonal antibodies have the advantage of unchanging specificity.

Human monoclonal antibodies (HUMAB) moreover avoid undesired immunoreactions of the recipient against heterologous monoclonal antibodies of murine origin, and appear to have a longer circulation time in the body.

The rabies virus is enveloped and of bullet-shaped morphology. The single stranded RNA of the virus is associated with a 55 kD nucleoprotein, the 190 kD "large transcriptase" molecule and the 38 kD nonstructural protein. The viral envelope contains a 26 kD matrix protein and a 67 kD glycoprotein. This glycoprotein is an important binding site for antibodies neutralizing the virus L. Lodmell, J. Virol., 61, 10: 3314-3318, 1987). The mechanism of neutralization is presumed to be an inhibition of the intraendosomal fusion step, i.e. an uncoating of the virus is prevented. Dietzschold et al.

(Virology, 161: 29-36, (1987)) describe the neutralization of a rabies virus by murine MAB against the 67 kD glycoprotein. Whilst a technique which has been established for a long time Kbhler and C. Milstein, Nature, 256, 495-497, 1975) allows murine monoclonal antibodies against certain antigens such as the glycoprotein of rabies virus to be established in a relatively simple way L. Lodmell, loc. cit.), it is extremely difficult to obtain human monoclonal antibodies for therapeutic use. Moreover it was not known whether such human monoclonal antibodies not only bind to the glycoprotein but can also neutralize infectious rabies virus and protect, in vivo, from the outbreak of the disease.

To obtain the human monoclonal antibody TW-1 against 3 rabies virus, which is described here, human peripheral blood lymphocytes of a healthy serum-positive donor were incubated with Epstein-Barr Virus (EBV). Supernatants of growing B-lymphocytes transformed by EBV were tested for rabies-specific antibodies in an ELISA. Specifically producing B-cell lines were fused with the mouse myeloma cell line SP2/0 in order to stabilize the antibody production. The resulting hybridoma were cloned several times and tested for specific production of antibodies of the IgG class. The process was modified by a known method Kozbor J. C. Roder, Immunol. Today, 4:72-79, 1983) and carried out as described in a different publication Hilfenhaus et al., Behring Inst. Mitt., 80:31-41, 1986). The selection of antibody-producing cells was, in the present case, carried out by testing the supernatants of the cultures in an ELISA on coating material containing rabies virus. The ELISA used is illustrated in more detail in Example 1.

It has been found that the human monoclonal antibody against rabies virus, TW-1, which is described here, reacts specifically with rabies-containing coating material (carried out with the Pitman-Moore, Fluri-LEP, CVS strains) but not with control material free of rabies virus in an ELISA. It has likewise been found that TW-1 does not bind to coating material containing control viruses, such as VZV, CMV, rubella, HSV or measles. TW-1 binds to fixed chicken fibroblasts infected with rabies virus but not to non-infected ones, since, after incubation of TW-1 with these cells and subsequent incubation with FITC-coupled antiserum against human immunoglobulin, it is possible to detect the fluorescence typical of rabies only on infected cells. Furthermore it has been found that TW-1 belongs to the IgG1 immunoglobulin class (lambda). After SDS gel electrophoresis of rabies antigen reduced by means of dithiothreitol (DTT) (carried out with the Pitman-Moore and Fluri-LEP strains), it was possible to find TW-1 reacting with a band around 67 kD in Western blots. It is possible to conclude from lr 4 comparisons of own data found and data in the literature H. Wunner et al., Reviews of Infectious Diseases, 4:771-784, 1988) that the protein recognized by TW-1 is the glycoprotein of rabies virus. This is confirmed by the observation that, after digesting the rabies antigen by means of endoglycosidase F before fractionating the antigen in the SDS gel, a reduction of the molecular weight of the protein marked by TW-1 can be observed in Western blots. Since endoglycosidase F cleaves off Nglycosidically linked sugar residues from proteins, it is possible to conclude from the data obtained that the band marked by TW-1 in Western blots is the glycoprotein of rabies virus. The cross-reaction of TW-1 with the rabies virus strains tested, Pitman-Moore, Fluri-LEP and CVS, indicate binding to a relatively cons(rved epitope within the glycoprotein.

The testing of whether TW-1 has the capability of neutralizing rabies virus was carried out by two different methods which are known per se. An "in vitro" fluorescence neutralization test was carried out as the first type of test. Antibody dilutions were mixed with active rabies viruses. After adding cells susceptible to rabies virus, the mixture was incubated for 24 hours then incubated with FITC-coupled antiserum specific for rabies and evaluated in a fluorescence invertoscope. Cultures which exhibit rabies-specific fluorescence are counted as positive. Since negative cultures only occur when rabies viruses are completely neutralized by added antibodies it is possible to examine an antibody for rabies-specific neutralizing activity by suitable dilutions and comparing with rabies-specific standard serum of known activity by means of this test. As the second neutralization test method, an experiment was carried out, in which, after mixing the antibody dilutions with infectious rabies virus and an incubation "in vitro", the mixture was administered intracerebrally to NMRI mice Atanasiu, Laboratory Techn. in Rabies, 3rd Edition, Editors M. M.

Kaplan and H. Koprowski, WHO (1973) 314-318). If active 5 rabies viruses are completely neutralized by added antibodies, ti. proportion of animals contracting the disease is smaller in this group than in the case of giving pure virus without previous admixture of antibody.

It has been found that rabies virus (CVS strain) is neutralized by TW-I both in the fluorescence neutralization test and in the neutralization test involving the administration to mice.

In order to demonstrate that giving TW-1 antibody can inhibit or prevent the outbreak of the disease in vivo in the case of an infection with rabies virus, protection experiments were carried out in NMRI mice. It has been found that TW-1 antibody can protect NMRI mice from the outbreak of the disease even 2 hours after the infection with rabies viruses. In comparison with a preparation from human sera containing antibodies against rabies (commercially available BerirabR, Behringwerke AG) significantly less protein is needed for the same neutralizing and protecting effect when giving TW-1. This is a significant advantage of TW-1.

In addition to the use as immunoprophylactic, the antibody, bound to a suitable carrier material, can also be used for the purification of rabies viruses or rabies virus glycoprotein by means of affinity chromatography processes. Apart from this, the antibody is suitable for validating the glycoprotein proportion in vaccine pre- I parations since rabies virus glycoprotein can be detected specifically, as is described in principle in the examples.

The invention therefore relates to an epitope on the 67 kD glycoprotein of rabies virus which is defined by the monoclonal antibody TW-1, and to polyclonal or preferably human monoclonal antibodies against the abovementioned epitope, very preferably TW-1 antibody.

The invention likewise comprises the use of 6 abovementioned antibodies for the preparation of passive vaccines, for diagnoses, for the purification of rabies viruses or rabies virus glycoprotein and for the validation of vaccine preparations. The invention is finally contained in the examples and the patent claims.

The hybridoma secreting the human monoclonal antibody TW-1 was deposited at the European Collection of Animal Cell Cultures (ECACC), Porton Down, Salisbury, UK on 26 February, 1990 with the No. 90022604 in accordance with the Budapest Convention.

Examples: The examples below are intended to illustrate the invention in more detail but do not restrict the invention.

For the experiments described in the examples, antibodies purified by affinity chromatography were used.

Example 1: Specificity of TW-1 antibodies in ELISA 96-well ELISA plates were coated with concentrates, which have been purified by sucrose gradient centrifugation and inactivated by means of p-propiolactone, of culture 21 supernatants of infected MRC-5 cells (Pitman-Moore strain) or infected chicken fibroblasts (Fluri-LEP strain), or with preparations of the corresponding noninfected cells as control, at room temperature for 24 hours. Dilutions of the antibody samples were incubated on the plate for 1 hour and unbound antibody was removed by washing. Bound antibodies were marked by incubating with a peroxidase-conjugated antibody (rabbit anti-human IgG, Behringwerke) for 1 hour. It was possible to detect human antibodies with a specificity for rabies virus via a peroxidase-catalyzed color reaction. In Fig. 1, the titration of TW-1 and BerirabR on the applied rabies antigens and the corresponding control antigens is shown.

If varicella zoster virus, cytomegalo virus, rubella viruses, herpes simplex virusl or measles viruses are 7 -7used for the coating, no reaction takes place. For this reason, it can be assumed that TW-1 binds to rabies virus with a high specificity. Since the binding curves of TW-1 against the rabies strains used have a very similar course, an approximately equal binding affinity of TW-1 against Fluri-LEP and Pitman-Moore can be assumed.

Example 2: Characterization of the binding epitope of TW-1 antibody in Western blots Purified rabies virus (Pitman-Moore strain) was fractionated in a 10% polyacrylamide gel according to Laemmli containing 1% SDS/50 mM DTT. After the separated antigen had been transferred onto nitrocellulose, incubation with TW-1 antibody, rabies-positive and rabies-negative human serum was carried out. The detection was carried out by biotin-coupled sheep anti-human immunoglobulin (Amersham), subsequent incubation with streptavidinperoxidase (Amersham) and developing with chloronaphtol (Merck). TW-1 antibody reacts with a band around 67 kD in Western blots (Fig. It can be concluded from literature data Wunner et al., loc. cit.) that this is the glycoprotein of rabies virus. This is confirmed by digesting the rabies proteins with endoglycosidase F (Boehringer Mannheim) before applying them to the SbS gel. The digestion was carried out according to the manufacturer's instructions. It was intended to observe a reduction of the molecular weight due to cleaving off of sugar residues from the glycoprotein. Such a reduction can be clearly seen for the 67 kD band in comparison with Sthe original antigen in Western blots after TW-1 antibodies have reacted, and this indicates a clycosylation of the protein recognized by TW-1 antibody. TW-1 antibody did not react with control antigens such as CMV, bovine serum albumin or human serum albumin.

8 Example 3: In vitro fluorescence neutralization test

(RFFIT)

The test was carried out in vitro by the method of Smith et al., S. Smith et al., Laboratory Techn. in Rabies, 3rd Edition, Editors M. M. Kaplan and H. Koprowsky, WHO (1973), 354-357) as microtest in microtiter plates Zalan, J. of Biol. Stand., 7:213-220, (1979)). Sample dilutions (antibody or PBS for the virus control) were mixed with infectious rabies virus. It was possible to deduce from the tests carried out (Fig. 2) that TW-1 antibody exhibits a neutralizing effect on the amount of virus used even at a concentration of about 0.14 ig/ml in this test. About 13.88 ig/ml of the reference serum used (horse) are required for the same effect. A human monoclonal antibody against Varizella zoster (VZV-6) of the same isotype which was used as a control did, in a comparable concentration, not have any effect on the infectiousness of the virus.

Example 4: In vitro neutralization test with inoculation of the antibody/antigen mixture into susceptible NMRI mice The test was carried out according to Atanasiu (loc.

cit.). NMRI mice with a weight of 20 g were used for the experiment. For the straining with virus, 60 LD 5 s per mouse were used. In an analogous way to Example 3, sample dilutions (antibody or controls) were initially mixed with active rabies virus in vitro and incubated. In order to monitor the neutralization, the mixture was then administered to NMRI mice intracerebrally. The mice were checked daily for 14 days and the number of surviving animals compared for TW-1 antibody, rabies-specific control serum, a VZV-specific hum,?b and the administration of pure virus. The corresponding data after an incubation time of 14 days are shown in Fig. 3. This makes clear that TW-1 antibody exhibits a clear neutralixation of rabies virus even at the low concentration of i ~P

II

9 0.08 pg/ml used, while the same concentration of VZV-6 does not have any neutralizing effect.

Example 5: Protection experiment The protection test was carried out using NMRI mice (weight: 20 g) susceptible to rabies virus. For the virus strain, 150 LD 5 were administered i.m. (CVS strain).

Dilutions of TW-1 antibody, commercially available BerirabR or of VZV-6 were administered i.v. 2 hours before or 2 hours after administration of active rabies virus.

In the experiment shown in Fig. 4, the same protection was achieved using 2.5 pg/ml IgG TW-1 antibody as when using 1.5 mg/ml polyclonal BerirabR. As control, VZV-6 was employed at double the concentration of TW-1 antibody.

The effect of VZV-6 was in the region of the virus control without administration of antibodies.

Legend for Fig. 1 (Example 1: Specificity of TW-1 antibody in ELISA 1 pg/ml TW-1 antibody and 100 pg/ml IgG Berirab (Behringwerke) were used as starting concentration.

Extinction was measured at 492 nm. The ELISA plates were coated with 0.4 IE/mi. A is extinction and Conc. is the concentration of the antibodies used.

TW-1 on rabies antigen TW-1 on control antigen o BerirabR on rabies antigen BerirabR on control antigen Legend for Fig. 2 (Example 3): In vitro neutralization test with fluorescence evaluation 4.4 pg/ml TW-1 antibody 1:100), 111.1 pg/ml 1:100) standard serum and 3.8 pg/ml VZV-6 1:100) were used as starting concentrations.

10 I.C. is infected cultures The antibody dilution (Dil.) is plotted on the abscissa TW-1 o rabies standard VZV-6 Legend for Fig. 3 (Example In vitro neutraliration test, evaluation in vivo 0.7 pg/ml TW-1 antibody 1:600), 18.5 g/ml BerirabR (Behringwerke AG) and 0.6 Ag/ml VZV-6 1:600) were used as starting concentrations.

S.A. is surviving animals The antibody dilution (DIL.) is plotted on the abticissa TW-1 o rabies standard VZV-6 Legend for Fig. 4: Protection experiments in vivo Fig. 4 A: administration of the antibodies 2 hours before administering the virus. Fig. 4 B: administration of the antibodies hours after administering the virus.

pg/ml TW-1 antibody, 1.5 mg/ml IgG BerirabR and pg/ml VZV-6 were used.

S.A. see above d.p.a. is days past administration

Claims (11)

1. An epitope on tho 67 kD glycoprotein of rabies virus, wherein the human monoclonal antibody secre- ted by the hybridoma TW-1 having the deposit No. 90022604 at the European Collection of Animal Cell Cultures (ECACC) reacts therewith.

2. A polyclonal or monoclonal antibody against the epitope as claimed in claim 1.

3. The human monoclonal antibody as claimed in claim 2, secreted by the hybridoma having the deposit No. 90022604 at the ECACC.

4. A process for the preparation of immunoprophylac- tics, comprising antibodies as claimed in claim 2 or 3 being used.

5. A process for detecting a rabies virus infection comprising antibodies as claimed in claim 2 or 3 being used.

6. A process for validating immunoprophylactics, com- prising antibodies as claimed in claim 2 or 3 being used.

7. A process for purifying rabies virus antigen, comprising antibodies as claimed in claim 2 or 3 being used.

8. A cell line or another expression system which produces antibody molecules as claimed in claim 2 or parts of these antibodies.

9. A cell line as claimed in claim 8, which results from transformation of human lymphocytes by Epstein- Barr virus and subsequent fusion with a myeloma cell. _1~ 12 The cell line TW-1 having the deposit No. 90022604 at the ECACC.

11. A polyclonal or monoclonal antibody as claimed in claim 2 or 3 as immunoprophylactic or diagnostic.

12. The use of antibodies as claimed in claim 2 or 3 for purifying rabies virus antigen, for diagnosis or for immunoprophylaxis. DATED THIS 28th day of February, 1991 BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS, 1st Floor, The Atrium, 290 Burwood Road, HAWTHORN. VICTORIA 3122.