JPH04501055A - Monoclonal antibodies to block infection of cells by the HIV-virus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Infection of cells with HIV-virus Monoclonal antibodies to block The present invention provides monoclonal antibodies, cell lines that produce the same, and HIM Regarding the use of this monoclonal antibody to block infection by viruses. do.

BIT-tile, X (also called ETLV-1- or LAv-1- ) is the etiological agent of acquired immune system deficiency disease (AIDS). of this virus Since its discovery, worldwide medical research has been carried out to develop drugs for the treatment of AIDS or for healthy humans. Attempts are being made to find possibilities to prevent the spread of infection. Already infected? Drug administration for people who are so-called EiIV-positive and already exhibit severe AIDS-syndrome. For example, AZT (azidothymidine) (which is effective against filthy The growth of this virus can be inhibited using substances such as (which block the formation of complementary DNA) Attempts are being made to prevent further spread and thereby avoid further deterioration of this patient's symptoms. Ru. However, such substances, which prevent the formation of intact DNA, It also has a negative effect on the cells, and the side effects that appear during the necessary long-term treatment are significant. stomach. Therefore, it cannot be said that an effective drug for eradicating AIDS has already been discovered. stomach. Furthermore, special attention should be paid to preventing infection among uninfected people. this Therefore, some laboratories have already developed a vaccine against this HIM-virus. The hand that manufactures it shows the strength. However, the measures that have been tried so far, In humans, the action of forming antibodies under the formation of so-called memory cells has not yet been successful. Not yet. This virus remains active after the body's primary immune response to the vaccine has weakened. The cells are not left unprotected after infection by this virus. The formation of memory cells that can induce an effective secondary immune response is necessary.

Therefore, the object of the present invention is to protect uninfected persons from infection by this HIM-virus. However, it may prevent the spread of the virus to healthy cells even in previously infected people. The goal is to provide the following. 5c by Kowalski et al. This virus surface protein g from Volume 237, pages 1351-1355 p120 is the so-called CD-4-protein (for example, helper T-97 7 present on the surface of globules, macrophages and other cells; Dalglelsh Nature, 3LLL (1984), 763; Klatz 767 pages of the same document by Mann et al., 5science by Me Dougal et al. 231.382 (see 198<S)), and therefore this virus It is known to measure the tissue specificity of staining. Other viruses with viral sheaths Similarly, after binding of pg120 to CD-4, virus entry into the cell is , which is facilitated by target cell-membrane sheath-mediated fusion in Vir2 cells.

By the way, the object of the present invention is the hybridome-cell line gcAcc 88050 502, which produced the monoclonal antibody 30F16H5, which also This is another object of the present invention. This monoclonal antibody is This protein binds to the CD-4 protein present on the surface of the target cell, and thereby Viral glycoprotein gp 120 to protein (BIVm simian immunodeficiency virus) Regarding the related monkey viruses, the binding of gp130) is This prevents Quills from entering the cell.

Obtaining monoclonal antibodies from hybridome cell lines is known per se. Do it in a method. That is, cells are cultured and a monoclonal antibody is applied, e.g. Obtained from the clear protein by Sepharose chromatography.

Another object of the invention is to increase the HI Monoclonal antibody 30P16H5 to block infection by M-virus is to use. The use of the antibodies according to the invention allows for example The first infection in a person is determined by the cD-4-protein associated with this HIM-virus. Therefore, it can be avoided to prevent the invasion of this virus. . However, in addition, daughter cells are susceptible to infecting other cells even after they have already been infected. This can help stop the spread of this virus, thereby stopping the virus infection. It can be stopped. Cells that have already been infected die after the virus multiplies, but This virus is no longer able to enter new cells. Therefore, H.I. After V-infection, the continual destruction of a small area is stopped as it causes onset of AIDS and animal death. do.

Therefore, another object of the invention is to prevent infection and/or susceptibility by the HIV-virus. This is a medicine to prevent the spread of this virus in the body after infection. The monoclonal antibody 30F16H5 according to the invention is contained in a pharmaceutically suitable preparation form. have In this case, customary pharmaceutical carrier substances and additives may be included.

Here, as an additive, for example, H IV - interferon or interferon that can contribute to rapid neutralization of the virus. -Loiki/ can be considered.

Another object of the invention is to treat the infection and/or post-infection of the body by the HIV-virus. The present invention provides methods for manufacturing pharmaceuticals that prevent the spread of this virus within the country. Monoclonal antibody 30F 16H5 may be prepared using conventional carrier substances and additives. This also makes it possible to obtain a pharmaceutically suitable application form.

A preferred application form of the medicament according to the invention is an advantageous solution suitable for intravenous injection.

That is, according to the present invention, monoclonal antibody 30F16H5 or hybrid By preparing the mu-cell line zchcc 88050502, H It has been successfully produced that drugs block the entry of IV-viruses. This means that In especially dangerous uninfected persons, it is necessary to prevent possible infection or, in already infected persons, to achieve complete immunity. The progress of the destruction of the epidemic system and the fatal consequences of the concomitant infection can be prevented.

The invention will be further explained in the following examples in conjunction with the drawings.

Figure 1 shows autoradiation of immunoprecipitation using various antisera and monoclonal antibodies. It shows the ophography.

FIG. 2 shows the results of a virus neutralization test using the antibody of the invention.

Figure 3 shows the results of virus neutralization tests using various monoclonal antibodies. There is.

Example 1 Production of hybridomes for immunization and production of monoclonal antibody 30F16H5 Cells of human CD4-positive helper T-lymphocyte clone 2C11 (Rmmric h & Kaufmann, Infection and Immunit75 1 (1986), 879) 4 x 106 to 8 x 10' pieces, 7 stitches apart each was injected intraperitoneally into 17 female BALB/C-W mice three times. Further immunization after 6 weeks I did this. The same amount of cells was applied intravenously the next day. 3 days after this last injection Afterward, the animals were sacrificed and the spleen cells were harvested using the methods of Cooler and Milstein. van K;;hlerund Milstone; Nature 25 6 (1975), 495) Yoshi, X63. λg8.655 myeloma cells and fusion (J, Immunol, 123 (1979) ), 1548).

After 10 to 14 days, remove the culture supernatant from the growth hyperdome and check the antibody activity. The properties were tested by in vitro functional tests. Antibody 30F16H5 intib , gLI8A (Emmrich et al., Eur, J. Immunol, 13 (1983), 273) as mouse-IgGi. hybrid A sample of the genome cells was deposited with the RCACC under accession number 88050502.

The specificity of this antibody 30F16H5 was systematically tested for cell-ELI8A, APAA It was measured by P-staining and lipofluorescence. Table 1 shows three different human cell populations. Representative results of the use of 30FISH5/μg7bt in binding to are shown.

Transformation of human B-cell lines (Immuno-biOIO of Teinitz et al. g7156 (1979) 41), a lymphoblastoid B-cell line was obtained and a homozygous Gen B - used as cell population. CD4- and CD8-'r- cells in a multi-step method isolated from human blood (Proc of Rmmrich et al., Natl, Aca d.

Sci, USA 83 (1986) 8298) eFor this purpose, first, Ficoll gradient solution (Ficoll gradient solution) for isolation of mononuclear cells dient) was used. Deplete adherent cells II on the plastic surface! KcS, % (depletiert), then T17 lymphocytes were obtained by rosette formation using stabilized sheep red blood cells. red blood cells After lysis of commercially available anti-CD 4 and anti-CD8 antibody, and the antibody-negative population was subjected to fluorescence activated cell sorting. Separation was performed using a Contamination with other cells was less than 1 for both populations. . Labeling with antibodies is performed using related methods (Gathings et al., I!ur, J.;

Immunnl, 7 (1970), 804). anti-maxu immunoglobulin-antibody coupled with isothiocyanate) It was carried out under the use of.

Table 1 shows that only CD4- cells can be recognized by antibody 30F16115. are doing. Other populations other than CD4 and CD8 on cells thus characterized 30F1611i5 recognizes the CD4-antigen, since no different antigen is known. Then it is concluded. This was confirmed by radioimmunoprecipitation experiments.

Table 1 1) FITC-labeled second antibody (Sheep anti-mouse with fluorescein -1g) Control 2) Antibody 30F16E5 and second antibody when used alone 3) Lymphoblast B-cell lineage Example 2 125I-1RRgp to CD-4-bearing cells with various sera or antibodies 130 O1a matching f)llll stop o inspection1t. GP130 European Patent Application No. 8810 Purified as described in No. 0191.1. This purified gp 130 , by Marlcvell and Fox K. Biochemistry (Biochemistry) 17. Described on page 4817 Iodination was carried out using Iodogsn as the oxidizing agent.

Next, we conducted a related combination study as follows: Mo1t-4-thin l! ! (Human lymphocytes @) 10' RPMI- without serum It was washed three times with medium (Fa, manufactured by GIBCO). The washed cells were washed with fetal serum. Serum skin 03111 in npMx (GIBCO) Q, 5iu containing 10ti or release in the presence or absence of antibody 0.0311j (equivalent to 1.5 μs of protein). Incubate for 2 hours at room temperature with radioiodinated gp 130 10' cpm. I started. Cells were then pelleted and washed 4 times each with serum-free RPMI. did.

This cell pellet was rinsed with Lyaiapuffer (Lisu HC-1). 10mM s fJ (8,0, NaC1140mQ DTT (dithiothlate ) i l1l) i (1PM8F (phenylmethylsulfonyl fluoride ) 1mM, NP-40 (Non1detP40, nonionic surfactant) Q, 510.10j and solubilized for 15 minutes at 4°C. This lysate Cleared at 10000.9 for 15 minutes. Supernatants Q and Q13i1 were added to reaction buffer ( 1 volume of rinse buffer, 2 volumes of phosphate buffered saline and ovalbumin o, 20 wkl ) Diluted with 0.08d. Next, naturally infected African green monkeys (a Add serum o, oos- obtained from frican green monkey) Incubate with this at 4℃ for 2 hours, then add 0.001 m of Hamabe serum. Immunoprecipitation reaction was carried out for 30 minutes by adding 100% protein-human 7aps 1da. . The precipitate was collected by centrifugation and washed with a high temperature washing buffer (Tris HCj 20ml). M5pH7.6, Na(J 500mM, KDTA 1mM%NF -40 0.55k, sodium deoxycholate 1t, sucrose 30t) once? Washing buffer with low salt content (Tris-yxca 10mM, pH 7.6, Ha ct (10 mM) twice. The precipitate was diluted with sample-buffer (Tris-Ect O, 05M% p) 16.8, Chaosthreitol 0.250M, Urea 8.0 00M, Sn22.300 and sample buffer 1-1 (saturated bromophenol) Addition of Le Blue solution) 0.05id and boiled for 4 minutes. Then cephalometric 0-03'It, 9-12 SO5DS-Ho! JT cells were subjected to electrophoresis in an amide gradient gel. electrophoresis After mobilization, the gel was dried and examined autoradiographically.

Figure 1 shows the autoradiography of such an immunoprecipitation; Each section a'-m indicates the CD-4-bearing cells in the presence of the following antisera or antibodies. Showing gp130 binding to the torso: a: no antiserum; b: gp 130 / x, t, al) / cFA2) Kusagi immune serum C: Kusagi pre-immune serum (non-immune serum) e: 2 weeks after immunization 17) gp 130 /KLHl)/ICFA4) Rhodorum-Immune Serum Serum G of Naturally Infected African Green Monkeys: Immunization h: 1st immunization i: OKT 4 monoclonal antibody (Firma 0rtho) j: MO Noclonal antibody Medac T4 (Firma Medae.

Hamburg) k: Monoclonal antibody Dakopatts T4-MK (Firm a　Dakopatts　1: Monoclonal antibody 30F16H according to the present invention 5m: Normal human serum (no antibody) 1) Schlisselloch snail (5chliissel-1ochschn ecke) hemocyanin 2) Complete Freund's adjuvant 6) Geltar aldehyde 4) Incomplete; gp 1 30 in Figure 1 in Table 2 - band radiation measurements are shown as inhibition (1) relative to the control.

Table 2 Blood serum suppression rate (*) gp 130Ar, a/cyh-rabbit serum 9a rabbit gull immune serum 2 Naturally infected green monkey serum 95 basics - 10 weeks after immunization gp13Q/KLa/ICFA 9 50KT 4 - MK 2 4 Medac T 4 - M K 8 9 Daklpatts T 4 - M K 9 130P1611!5 9 5 From now on, monoclonal antibody 30F16H5 of the present invention, 2>KMolt - 4-The binding of gp130 to the CD-4 protein of cells was detected using other monoclonal It can be seen that the inhibition is significantly greater than that of Le.

Example 3 Preparation of the antiserum used in Example 2 Anti-gp130 antiserum in Rhesusaffen Manufactured by European Patent Application No. 8 8 1 0 0 1 9 1. stated in the specification of item i I did it as it was written.

Rabbit antiserum was produced as follows: Purified gp13030μs was purified by KIJ ( Mixed with snail hemocyanin), complete 70 indian ali It was emulsified in Juvans (CFA). Pour half of this emulsion into the rabbit's abdominal cavity. Injected inside. Four weeks later, the other half was injected intramuscularly. Blood antiseptics used above The supernatant was removed 2 weeks after the second injection.

Example 4 Virus-neutralization test for antibodies according to the present invention This neutralization test was performed using solder (Haraaa ) et al. Immun.

MetL. 9 2, (1986), pp. 77-181, J. Handa et al. C11 n. Mi. crobiol. 2 2, (1985), pages 908-911 It was carried out as described. For this, nine MT4-cells were transformed with HTLV. (which is very sensitive with respect to V-cell pathogenesis) was used (Hara da et al. 5science 2 2 9, (1985), pp. 566-566). cormorant Decreased viral infectivity as a marker of cell proliferation (3H-thymidine absorption) This was confirmed by measurement. First, from the antibodies, 20 ml of inactivated fetal bovine serum and R@pes Click (C11ck)-RFMr (Fa. Pre-dilution of 1:10 to 1:1280 in Biochrom Inc.) made. Each predilution (100 μt) of antibody was then injected into the same amount of infectious mixed with the virus. This infectious viral child is a (3H)-thymidine of uninfected cells. It is defined as the amount of virus that can reduce the introduction of viruses by more than 90%. this From the mixture, 50μt was added to the same volume of MT4-cell suspension (6 x 10b cells per cell). ) in a microtiter plate with 96 wells. antibody final Dilution ratios ranged from 1:40 to 1:5120. Cells were treated with fetal serum 20% and H Click-RFMr (Fa.

Biochrom) at 70° C. in a 5% CO atmosphere. 6th day Afterwards, 100 μt of fresh medium was added to the cells. Four days after infection, (5H)- Chimidji/0.1 μC was added. After 20 hours, cells were transferred onto glass fiber filters. and washed with 10ts trichloroacetic acid. The radioactivity of the filter was measured.

All experiments were performed in triplicate. Figures 2A-D show the HIV-1-IB (second A), HIM-2-ban (Fig. 2B), SIVmac (Fig. 2C), and According to the present invention regarding infection by SI'V agm-TYO-7 (Figure 2D) It shows the neutralizing effect of antibody 30F 16H5 at various dilutions.

From these, the antibody of the present invention can inhibit HIV infectivity up to a dilution of 1:1600. , and it was confirmed that the infectivity of HIM I could be suppressed by 75% at a dilution of up to 800. Comparison of virus neutralizing effects of various antibodies Here, too, first of all is Click-RPMI (Fa, Biochrom). that 25 μt of each antibody dilution was added to the MT4-cell suspension (1 t/6×1 of our cells). 0') for 1 hour at 37°C. Final dilution of antibody 1:60~1:! It has spread to 1840. The cells treated in this way were then Centrifuge at 1500 rpm in a centrifuge (Haereus Manifuge) Click-R with 25+ nM of isolated fetal bovine serum and agpgs PMT (Fa, Biochrom) 100μ! The corresponding Hn'-1 in - lb infectious dose at 37°C in a 5ToCOz-atmosphere. Other treatments was similar to Example 4. Figure 3 shows the neutralizing effect of various commercially available antibodies on the antibody of the present invention. It is shown in comparison.

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Claims (5)

[Claims] 1. Hybridoma cell line ECACC 88050502. 2. The hybridoma cell line ECACC 88050502 is obtained from Clonal antibody 30F16H5. 3. by HIV- or SIV-viruses of cells with CD4-protein on their surface. Using monoclonal antibody 30F16H5 to block infection. 4. Monoclonal antibody 30F16H5 is optionally carried with conventional pharmaceutical carriers and HIV- or SIV-viral susceptibility characterized in that it contains together with additives and additives. drugs that prevent infection and/or prevent the spread of this virus within the body after infection. 5. Monoclonal antibody 30F16H5 optionally with conventional carrier substances and additives HIV- or SIV, with the aim of making it a pharmaceutically suitable form for female use. - prevent infection by the virus and/or spread of this virus within the body after infection; A method for manufacturing drugs to prevent