AU629847B2 - Monoclonal antibody for inhibiting cell infection by hiv viruses - Google Patents

Description

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0PJ DATE 23/03/90 APPLN. I D 4I0702 89 A0JP DATb 2h 4I! 0 PCT NUMBER PCI/EP89/00986 INTERNAT~u.- ALE .Z AE E P T INTERNATIONAL USAN U~~M BIJIDES PATENT\VESN PT (51) lnternati6riae Patentklassifikation 5'(I nentoaeVefetihnsumr WO 90/02199 CI 2P 21/08, A61 K 39/395 Al (43) Internationales //C12N 5/20 Veriffentlichungsdatum: 8. Mairz 1990 (08.03.90) (21) Internationales Aktenzeichen: PCT/EP89/00986 (74) Anwijte: WEICKMANN, H. usw. MWhIstrage 22, D- 8000 M~inchen 80 (DE).

(22) Internationales Anmeldedatum: 22. August 1989 (22.08,89) (81) Bestimmungsstaaten: AT (europaisches Patent), AU, BE PrioritOtsd aten: (europ~isches Patent), CH (europgisches Patent), DE P 38 28 582.7 23. August 1988 (23.08.88) DE (europtiisches Patent), DK, Fl, IR (europ~isches Patent), GB (europ~isches Patent), IT (europiiisches Patent), JP, LU (europ~isches Patent), NL (europ~iisches (7 1) Anmelder (ftir alle Besirnungssaaen ausser US): MAX- Patent), NO, SE (europaiisches Patent), US.

PLANC K-G ESELLSC HAFT ZUR FORDERUNG DER WISSENSCRAFTEN E.V. [DE/DE]; Bunsenstra- S~e 10, D-3400 Gbttingen DEUTSCHES PRIMA- Verdffentlicht TENZ ENTRU NI G ES ELLSC RAFT MB H [D E/ DE]; Alit internauionalemn Recherchenberich.

Kellnerveg 4, D-3400 Gbttingen (DE).

(72) Erfinderund Erfinder/Anmelder (nur~flir LIS) ENINIRICH, Frank [DE/ DE]: H~henweg 4, D-8521 Braeuningshof HUNS- MANN, Gerhard [DE/DE]; Holundersteg 9, D-3400 G~ttingen LUKE. Wolfgang [DE/DE]; David- Hilbert-Strage 2, D-3400 Gbttinoen (DE).

(54)Title: MIONOCLONAL ANTIBODY FOR INHIBITING CELL INFECTION BY HIV VIRUSES (54) Bezeichnung: NIONOKLONAL!- ANTIKORPER ZUR INHIBIERUNG DER INFEKTION VON ZELLEN DURCH

HIV-VIREN

(57) Abstract The monoclonal antibody 30F16H5T 4 -MK, which can be obtr~ined from the hybridoma cell line ECACC 88050502. is used to inhibit the infecuion by HIV and SIV viruses of cells with a CD4-protein on their surface. A drug For preventing infection by HIV viruses and/or the spreading of viruses in the body after infection contains the monoclonal antibody 30F16H5, optionally with the usual pharmaceutical carrier substances and additives.

(57) Zusammenfassung Der monokionale Antik~rper 30F16H-5T 4 -MK, erhilltlich aus der Hybridom-Zellinie ECACG 88050502, wird zur Inhibierung-der lnfektion von Zellen, welche emn CD4-Protein auf ihrer Oberflache aufweisen, durch HIV- und SIV-Viren verwendet. Fin Arzneimittel zur Verhiltung einer Infekfion durch HIV-Viren oder/und der Ausbreitung von Viten im nach erf'olgter Infektion enthillt den monokionalen Antikbrper 30F7161-15, gegebenenfalls mit tiblichen pharmazeutischen Trilger- und Zusatzstoffen.

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ri The invention concerns a monoclonal antibody, a cell line producing it, as well as the use of the monoclonal antibody for the inhibition of the infection of cells by the HIV virus.

The HIV virus, also called HTLV-III or LAV-1virus, is the aetiological agent of the acquired immune deficiency AIDS. Already since the discovery of this virus, worldwide in medical research a medicament has been sought for the treatment of AIDS or to find a possibility for the prevention of the infection of healthy humans. In the case of medicinal treatment of already infected, so-called HIV-positive persons who in turn already show severe AIDS symptoms, it has been attempted to prevent a spreading of the viruses with substances, such as e.g. AZT (azidothymidine), which block the formation of DNA complementary to the viral DNA and thereby to avoid a further deterioration of the state of the patient.

However, such substances which prevent the formation of intact DNA also have a negative effect on healthy cells and the side effects arising in the case of the necessary long-term treatment are considerable. Therefore, it cannot be said that an effective medicament for the combating of AIDS has already been found.

Furthermore, there must especially be kept in view the prevention of the infection of still non-infected i''

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3 persons. For this purpose, there are already projects in some laboratories to produce vaccines against the HIV virus. However, with the previously tested strategies, it has not yet been possible to bring about in humans the formation of antibodies with formation of so-called memory cells. The formation of the memory cells is necessary in order that, after subsidence of the primary immune reaction against the vaccine, the body does not again remain unprotected against the virus but rather, after infection by the virus, these cells can initiate an effective secondary immune reaction.

Therefore, it is the task of the present invention to provide a possibility not only to protect non-infected persons against an infection by the HIV virus but also, in the case of infected persons, to prevent a spreading of the virus to still healthy cells. It is already known from Kowalski et al., Science Vol. 237, pages 1351 to 1355, that the viral surface protein gpl20 binds to the so-called CD-4 protein which is present on the surface of, for example, helper T lymphocytes, macrophages and other cells (Dalgleish et al., Nature, 312, (1984), 732; Klatzmann et al., ibid., page 767; McDougal et al., Science 231, 382 (1986)) and thus determines the tissue specificity of the viral infection. Analogously to other viruses with a virus sheath, after the binding 1 i' 'i i n tyll

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of gpl20 to CD-4, the virus entry into the cell is facilitated by a sheath-mediated fusion of the viral and target cell membranes.

An object of the invention is now a hybridoma cell line, ECACC 88050502, which produces a monoclonal antibody 30F16H5, which, in turn, is a further subject of the Application. This monoclonal antibody binds to the CD-4 protein which is present on the surface of the target cells of the HIV viruses and thereby prevents the binding of the viral glycoprotein for SIV simian immunodeficiency virus, a monkey virus closely related to HIV) to the protein and thereby the virus entry into the cell.

The obtaining of the monoclonal antibody from the hybridoma cell line takes place according to per se known methods, namely, the cells are cultured and the monoclonal antibodies obtained e.g. from the cell supernatant by protein A sepharose chromatography.

A further subject of the invention is the use of the monoclonal antibody 30F16H5 for the inhibition of the infection of cells which have a CD-4 protein on their surface by HIV viruses. More particularly, there is provided a method for inhibiting HIV or SIV infection of cells which have a CD4 protein on their surface by contacting with said cells an effective amount of monoclonal antibody 30F16H5, as hereinbefore defined. By means of the use of the antibody according to the invention, a first infection e.g. in the case of especially endangered persons, can be avoided by blocking of the CD-4 protein for the HIV virus and thus the prevention of 920817,ejhdat093,40702.et,4

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-P f P~a-: i the penetration of the virus. Furthermore, however, even after infection has already taken place, the spread of the virus by infection of further cells by daughter viruses can be prevented, whereby the virus infection comes to a halt. The already infected cells die off after virus multiplication but the virus can no longer penetrate into new cells. Therefore, the progressive destruction of the T cells ceases which, after an HIV infection, leads to the AIDS form of the disease and to death.

Therefore, a further subject of the invention is a medicament for the prevention of an infection by HIV viruses and/or of the spread of the viruses in the body after infection has taken place which contains the monoclonal antibody according to the invention 30F16H5 in a pharmaceutically suitable form of administration. There can hereby be included further usual pharmaceutical carrier and additive materials.

As additive materials, there are here conceivable, for example, interferons or interleukins, which can contribute to an activation of the immune system and thus to the quicker neutralisation of the HIV viruses circulating in the blood.

A further subject of the invention is a process 25 for the preparation of a medicamient for the prevention of an infection by HIV viruses and/or of the spread of the viruses in the body after infection has taken

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6; a a :Ca 6 place, which is characterised in that one brings the monoclonal antibody according to the invention 30F16H5, possibly with usual carrier and additive materials, into a pharmaceutically suitable form of administration.

Suitable forms of administration for the medicament according to the invention are preferably solutions which are suitable for intravenous injection.

Thus, according to the invention, the making available of the monoclonal antibody 30F16H5 or of the hybridoma cell line ECACC 88050502, makes it possible to make available an agent for the blocking of the penetration of HIV viruses into their target cells. This can prevent a possible infection above all in the case of endangered non-infected persons or, however, also prevent the progressive destruction of the whole immune system and thus the lethal consequences of the infection in the case of already infected persons.

The following Examples further explain the invention in conjunction with the drawings.

i Fig. 1 shows the autoradiograph of an immune precipitation with various antisera and monoclonal antibodies; Fig. 2 shows the results of a virus neutralisation test with the antibody according to the invention; 11 ii 7 Fig. 3 shows the results of a virus neutralisation test with various monoclonal antibodies.

Example 1.

Immunisation and production of the hybridomas for the preparation of the monoclonal antibody 30F16H5: 4 x 106 to 8 x 106 cells of the human CD4positive helper T lymphocyte clone 2C11 (Emmrich Kaufmann, Infection and Immunity 51 (1986) 879) were injected three times, in each case in an interval of 7 days, into female BALB/c mice intraperitoneally.

A further immunisation took place after three weeks.

On the following day, the same cell amount was administered intravenously. 3 days after the last injection, the animals were killed and the spleen cells fusioned with X63.Ag8.653 myeloma cells (Kearney et al., J. Immunol. 123 (1979) 1548) according to the method of K6hler and Milstein (Nature 256 (1975) 495).

After 10 to 14 days, culture supernatant was taken from the growing hybridomas and the antibody activity thereof investigated in vitro in the functional test. The isotype of the antibody 30F16H5 was determined by means of ELISA (Emmrich et al., Eur.

J. Immunol., 13 (1983) 273) from mouse IgGl. A sample of the hybridoma cells was deposited with the ECACC under the deposition number 88050502.

The specificity of the antibody 30F16H5 was demonstrated methodically by cell ELISA, APAAP 1 1 j' 1 T I- 8 coloration and cytofluorometry. In Table 1 are illustrated three representative results of the use of 1 pg./ml. 30F16H5 in the case of the binding to three different human cell populations. A lymphoblastoid B cell line was produced by transformation of human Blymphocytes (Steinitz et al., Immunobiology 156 (1979) 41) and used as homogeneous B cell population. CD4 and CD8 T cells were isolated from human blood via a multi-step process (Emmrich et al., Proc. Natl. Acad.

Sci. USA 83 (1986) 8298). For this purpose, a ficoll density gradient was first used for the separation of the mononuclear cells. Adherent cells were depleted on plastic surfaces and then T lymphocytes were obtained by rosetting with stabilised sheep erythrocytes. After lysing of the erythrocytes, the T cells were labelled with commercially available anti-CD4 and anti-CD8 antibodies and separated as antibody-negative population with a fluorescence-activated cell sorting apparatus. The contamination with other cells lay, for both populations, below The labelling with antibodies took place according to appropriate methods (Gathings et al., Eur. J. Immunol. 7 (1977) 804) with use of an anti-mouse immunoglobulin antibody which was coupled with FITC (fluorescein isothiocyanate).

Table 1 shows that only CD4 cells are recognised by the antibody 30F16H5. Since no further populationdifferent antigens apart from CD4 and CD8 are known 1 I 9 on the so characterised cells, the interference is definite that 30F16H5 recognises the CD4 antigen.

This was confirmed by radio-immune precipitation experiments.

Table 1 cell fluorescence- average fluorescencel population positive cells intensity 1) 2) 2) 16H5 16H5 2) 3) LBL 2.3 3.4 48 CD8 T cells 3.2 3.5 CD4 T cells 3.8 84.6 658 1) control in the case of sole use of an FITC-labelled second antibody (goat anti-mouse Ig with fluorescein) 2) antibody 30F16H5 and second antibody 3) lymphoblastoic B cell line Example 2 Investigations of the inhibition of the binding of 1 2 5 I-labelled gpl 3 0 to cells having CD-4 by various sera or antibodies. Gpl30 was puridied as described in European Patent Application 88 100 191.1. The purified gpl30 was iodised with iodogen as oxidation agent as described by Markwell and Fox (1978), Biochemistry 17, p. 4817. Binding studies were then carried out herewith as follows: 6 molt-4 cells (human lymphoma cells) were washed three times with RPMI culture medium (firm GIBCO) without serum. The washed cells were incubated for 2 hours at room temperature with 10 cpm of the radio-iodated gpl30 in the presence or absence of 0.03 ml. serum or 0.03 ml. (corresponds to 1.5 pg.

protein) of antibody in 0.50 ml. RPMI (firm GIBCO), containing 10% foetal calf serum. The cells were then pelletised and washed four times with, in each case, 1 ml. RPMI without serum. The cell pellet was resuspended in 0.10 ml. lysis buffer (10 mM Tris-HCl, p1l 8.0, 140 mM NaC1, 2 mM MgCl 2 1 mM DTT (dithiothreitol), 1 mM PMSF (phenylmethylsulphonyl fluoride), NP-40 (Nonidet P 40, non-ionic detergent)) and solubilised for 15 minutes at 4°C. The lysate was clarified at 10,000 g for 15 minutes. 0.08 ml. of the supernatants were diluted with 0.08 ml. of reaction buffer (1 volume of lysis buffer, 2 volumes of phosphate-buffered common salt solution and 0.20 mg./ml. ovalbumin). 0.008 ml. of a serum obtained from a naturally infected African green monkey were t then added thereto and incubated therewith for 2 hours at 4°C. Thereafter, an immune precipitation was carried out by addition of 1 mg. protein A sepharose per 0.001 ml. of the added serum for minutes. The precipitates were collected by centri- S. fuging and washed once with wash buffer with high -i l c~ 11 salt content (20 mM Tris-HCl, pH 7.6, 500 mM NaCI, 1 mM EDTA, 0.5% NP-40, 1% sodium deoxycholate, sucrose) and twice with wash buffer with low salt content (10 mM Tris-HCl, pH 7.6, 10 mM NaCI). The precipitates were taken up in 0.05 ml. of sample buffer (0.050 M Tris-HC1, pH 6.8, 0.250 M dithiothreitol, 8.000 M urea, 2.300% SDS and addition of 0.005 pl. of a saturated bromophenol blue solution per 1 ml. of sample buffer) and boiled for 4 minutes.

The sepharose was then pelleted at 10,000 g for minutes and 0.03 ml. of the supernatants were subjected to electrophoresis in a 9 to 12% SDS polyacrylamine gradient gel. After the electrophoresis, the gel was dried and an autoradiography carried out.

Fig. 1 shows the autoradiograph of such an immune precipitation, whereby the individual columns a to m give the binding of gpl30 to CD-4-carrying cells in the presence of the following antisera or antibodies: a without antiserum b gpl30/KLH 1

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2 rabbit immune serum c rabbit preimmune serum (non-immune serum) d gpl30/GLA3)/KLH 1

/ICFA

4 rhesus immune serum, two weeks after immunising Se gpl30/KLH 1

/ICFA

4 rhesus immune serum, two weeks after immunising Sj f serum of a naturally infected African green monkey g serum as d but 10 weeks after immunising n v N .0 i 7 1 1 1 1 i i

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t 12 h serum as e but 10 weeks after immunising i OKT4 monoclonal antibody (firm Ortho) j monoclonal antibody Medac T4 (firm Medac, Hamburg) k monoclonal antibody Dakopatts T4-MK (firm Dakopatts) 1 monoclonal antibody 30F16H5 according to the invention m normal human serum (without antibody) 1) haemocyanin of the keyhole limpet 2) complete Freund's adjuvant 3) glutaraldehyde 4) incomplete Freund's adjuvant In Table 2 are given the results of the measurement of the radio-active radiation of the gpl30 bands of Figure I as inhibition with regard to a control.

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1I 13 Table 2 serum inhibition rabbit immune serum rabbit pre-immune serum 2 rhesus immune serum, two weeks after immunising rhesus immune serum 53 two weeks after immunising 10 serum of a naturally infected African green monkey gpl30/GLA/KLH/ICFA 10 weeks after 96 base immunising 10 weeks after base 93 immunising OKT4-MK 24 Medac T4-MK 89 Dakopatts T4-MK 91 30F16H5 It is to be inferred herefrom that the monoclonal antibody 30F16H5 according to the invention inhibits the most strongly the binding of gpl30 to the CD-4 protein of the molt-4 cells in comparison with the other monoclonal antibodies.

Example 3 Preparation of the antisera used in Example 2 The preparation of the anti gpl30 antisera in the rhesus monkey took place as described in European Patent Application 88 100 191.1.

14 The rabbit antiserum was prepared as follows: pg. of the purified gpl30 were mixed with KL (haemocyanin of the keyhole limpet) and emulsified in completL Freund's adjuvant (CFA). Half of this emulsion was injected subcutaneously into a rabbit.

After 4 weeks, the other half was injected intramuscularly. The here used antiserum was taken 2 weeks after the second injection.

Example 4 Virus neutralisation test of the antibody according to the invention The neutralisation test was carried out according to Harada et al., J. Immun. Meth., 92 (1986) 77-181, Harada et al., J. Clin. Microbiol. 22, (1985), 908-911.

HTLV I-trensformed MT4 celxs were used herefor which are very sensitive for HIV I cytopathogeneity (Harada et al., Science 229 (1985), 563-566). The reduction of the viral infectivity was determined by measurement of the 3 H)-thymidine take-up as label for the cell multiplication. First, from the antibody, predilutions were prepared of 1:10 to 1:1280 in Click-PMI S: (firm Biochrom) with 20% inactivated foetal calf serum and 25 mM Hepes. The particular predilutions of the antibody (100 p1.) were then mixed with an equal volume of the infectious virus dose. The infectious virus is defined as the amount of virus which can reduce the 3 H)-thymidine incorporation of non-infected cells by more than 90%. Of this mixture, 50 pl. were then added to an equal volume of an MT4 cell suspension (6 x 105 cells per ml.) in a microtitre plate with 96 cups. The end dilutions of the antibody then extended from 1:40 to 1:5120. The cells were cultured in Click-RPMI (firm Biochrom) with foetal calf serum and 25 mM HEPES at 37 0 C. in a

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2 atmosphere. After 3 days, the cells were provided with 100 pl. of fresh medium. 4 days after infection, 0.1 pCi 3H)-thymidine were added to the cells. hours later, the cells were collected on glass fibre filters and washed with 10% trichloroacetic acid.

The radioactivity of the filter was determined. All experiments were carried out three times. Fig. 2 A-D shows the neutralising effect of the antibody 30F16H5 according to the invention in various dilution for the infection of cells by HIV-1-IIIB (Fig. 2A), HIV-2-ben (Fig. 2B), SIVmac (Fig. 2C) and SIVagm-TYO-7 (Fig. 2D).

It can be seen therefrom that the antibody according to the invention is able to suppress the infectivity of HIV I down to a dilution of 1:1600 and of HIV II down to a dilution of 1:800 to Example Comparison of the virus-neutralising action of various antibodies Here, too, there were first prepared pre- 9' I 16 dilutions of the antibody of 1:10 to 1:1280 in Click-RPMI (firm Biochrom) with 20% foetal calf serum in 25 mM HEPES. 25 pl. of the antibody dilution in question were incubated with 50 pi. of an MT4 cell suspension (6 x 106 cells per ml.) for one hour at 37 0 C. The end dilution of the antibody then extended from 1:30 to 1:3840. The so treated cells were then centrifuged off for 10 minutes at 1500 r.p.m. in a centrifuge (Haereus Minifuge) and cultured with the corresponding HIV-1-IIIb infectious 10 does in 100 pl. Click-RPMI (Firm Biochrom) with 20 foetal calf serum and 25 mM Hepes at 37 0 C. in a 5% CO 2 a* atmosphere. The further manner of proceeding corresponded to Example 4. Fig. 3 shows the neutralising effects of various commercially available antibodies in comparison with the antibody according to the invention.

Microorganism Deposit: A hybridoma cell line was deposited with the Public Health Laboratory Service, Porton Down, Salisbury, Wiltshire SP4 OJG, England, on 5th May, 1988 and was feel accorded Accession No. 88050502.

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Claims (4)

1. Hybridoma cell line ECACC 88050502.

2. Monoclonal antibody 30F16H5 as hereinbefore defined.

3. A method for inhibiting HIV or SIV infection of cells which have a CD4 protein on their surface by contacting said cells with an effective amount of monoclonal antibody 30F16H5, as hereinbefore defined.

4. A medicament for the prevention of an infection by HIV or SIV viruses and/or of the spread of viruses in the body after infection has taken place, comprising monoclonal antibody 30F16H5 as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents and/or additive materials. A process for the preparation of a medicament for the prevention of an infection by HIV or SIV viruses and/or of the spread of the viruses in the body after infection has taken place, characterised in that one brings the monoclonal antibody 30F16H5, as hereinbefore defined, possibly with usual carrier and additive materials, into pharmaceutically suitable form of administration. i DATED this 20th day of August, 1992 Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V. and Deutshes Primatenzentrum Gesellschaft mbH By Their Patent Attorneys DAVIES COLLISON CAVE 920820,eihdat.093,40702.et, 17 \LZ