JPH06500323A - How to treat viral infections - 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] How to treat viral infections field of invention TECHNICAL FIELD The present invention relates to a method for treating viral infections in mammals. Anti-TNFIJ Gant alone or in combination with tumor necrosis factor (TNF) alpha This includes administering either one at the same time. The above-mentioned ligand is TNF properties such that the biological activity of TNF is altered when its ligand binds to There are signs. The invention also relates to compositions for treating viral infections.

Background of the invention Tumor necrosis factor (TNF) alpha is derived from Bacillus carme, teguerin (ijB Corynebacterium verbum (Niji μ-ku) First observed in the serum of experimental animals presensitized by LPS; It is a product of activated macrophages.

Following systematic administration of TNF, hemorrhagic necrosis was induced by TNF in vitro. The murine tumor produced cytolytic or proliferative effects on the abscess cell lineage. observed in some transplantable abscesses.

In addition to its host-protective effects, TNF has been shown to be effective in sepsis, cachexia and cerebral malaria. It is included as an alternative causative agent for pathological conditions. PO against TNF Passive immune production in mice in conjunction with reclonal rabbit serum has been shown to reduce toxicity. The lethal effects of LPS endotoxin can be counteracted when the starting drug is administered prior to infection. This is shown by protecting mice.

The gene encoding TNF is a potential cancer therapeutic that should be evaluated. It has been cloned to recognize the efficacy of Mo/Kine. TNF for cancer patients In stage I clinical trials injected with Side effects such as hypocytosis, cytopenia, hepatotoxemia, kidney damage and hypertension have been reported. ing. The clinical use of TNF with these very serious side effects has These are expected in view of the effects of TNF on knowledge, and some of them are listed in Table 1. Described.

Table 1 of · mono-anti-neoplastic - anti-viral Mono-anti-parasitic action Cytotoxic effects of intestinal tumor cells Pyrogenic activity Inhibition of angiogen Ic-activated riboprotein lipase Revitalization of baseball stadiums osteoclast activation Endothelial attraction, mononuclear cells and tumor cells Secondary coagulant activation Attraction of surface antigens on endothelial cells IL-6 attraction Attraction of c-myc and c-fos Attraction of EGF receptors IL-1 attraction Induction of TNF synthesis Induction of GM-C3F synthesis Increased prostaglandino and collagenase synthesis acute phase protein c3 Of particular importance are the effects that occur as a result of TNF activation of endothelial and also peripheral blood mononuclear cells. This is the activation of coagulation. Disseminated intravascular coagulation is toxic/acute and cancer of the intestine. cancer, pancreatic, prostate, lung, breast and ovarian carcinoma, melanoma, acute leukemia, myeloma, It has been associated with many carcinomas, including myeloproliferative syndromes and myelosporetic leukemia.

If no clear alterations in TNF activity remain, such as degenerative activity in abscesses, coagulation Undesirable effects such as activation of cancer cells can be eliminated or masked for further treatment of cancer. This will pave the way for greater effectiveness. Complete disappearance of TNF activity is a treatment for toxic 7gy be praised for its success.

Through the use of positionally specific antibodies (both polyclonal and monoclonal) An increase in hormonal activity can occur as a result of uncoupling of mon activity. (As Tong et al., Mo1. lsmunolL, 435 (1986)). until today Some attempts have been made to assign antigenicity or to NF has been created that acts on a special region of the molecule. The ratio of effects on the same area The project will advance the development of monoclonal antibodies (MAbs) and other ligands for therapeutic use. It will be possible. Amino acids 1 to 15 of polyclonal antibodies are reported to block the receptor of He1aR19 cells binding to (Sokar et al., PNAS IL 8829 (1987)) Recognition of adaptive epitopes on TNF is unclear and inhibition in vitro TNF cytotoxicity has been shown (Brinobuman and Agawel et al., Hybrido Mat, 4H (1987)) However, these effects on other TNF activators The effects of antibodies are unknown.

Description of the invention The inventor has produced a panel of monoclonal antibody activities against human TNF. The anti-tumor effects of TNF (both in vitro and in vivo), the binding of receptors, activation of coagulation (both in vitro and in vivo) and their localization. It has characteristics related to the effect of clarification of topological singularity. This invention led by the inventor The approach suggests that different topological regions of TNF-alpha may account for differences in activity. This is indicated by interlocking. This work is authorized under the Patent Cooperation Treaty. Simultaneous continuation filing based on application dated August 7, 1990 with Stralia as receiving Office The details of this application are disclosed in the application, and the disclosure of this application is hereby incorporated by reference. It is concrete.

The present inventors have combined their efforts to provide specific anti-TNF ligands useful for anti-viral therapy. They have made surprising discoveries through the administration of TNF. In addition, it has a unique anti-T Administration of NF ligand alone indicates that the ligand binds well to endogenous TNF. It was believed that the antiviral therapy could provide efficacy in antiviral therapy, and A similar effect was also provided when anti-TNF was administered in combination with TNF. Ru. Administration of this anti-TNF ligand alone resulted in elevated endogenous levels of TNF. would be the preferred method of treatment in the disease state.

Summary of the invention Accordingly, a first aspect of the invention provides for administering anti-TNF ligands alone or to a mammal. in mammals, including administration of either one in combination with NF. In the method of treating a viral infection, when the ligand binds to TNF, ffT The expression of the endothelial coagulant activity of NF is blocked and the anti-viral activity of TNF is inhibited. A method for treating viral infections characterized by the fact that the sex is unaffected or increased. Rinaru.

In a preferred embodiment of the invention, when the ligand binds to TNF, Binding to receptors on endothelial cells is inhibited; abscess fibrils/precipitates and TNF The tumor regression activity of TNF was increased; its cytotoxicity was not affected, and the abscess level of TNF was A further feature is that receptor binding activity is unaffected or increased.

A further preferred embodiment of the invention is that the ligand has a topological structure of residues 1 to 18. The naturally occurring biological activity of TNF epitopes defined by It is characterized by substantially inhibiting binding to sexual ligands.

In a still further preferred embodiment of the invention, the topological region of residues 1-30.11 7-128 and 141-135, more preferably topological region 1 of said residues -26. Epidemiology of TNF defined by 117-128 and 141-153 to substantially inhibit binding of the tope to naturally occurring biologically active ligands. The ligand is bound to TNF in such a manner that the ligand binds to TNF. Such an arrangement region is shown in the topology in Figure 16. expressed geometrically.

A second aspect of the invention provides anti-TNF IJ Gant alone or TNF in mammals. in mammals, including administration of either one in combination with NF. In the method of treating a viral infection, the ligand comprises residues 1 to 18 of human TNF. A method for treating viral infections characterized by binding to.

A third aspect of the invention provides for administering an anti-TNF ligand alone or in combination with TNF to a mammal. Viral activity in mammals, including administration of either one in combination In the method of treating an infection, the ligand comprises residues 1-30, 117-128 and 14. It is characterized by binding to human TNF within the topological region of 1-153. It consists of a method for treating viral infections.

In a preferred embodiment of this aspect of the invention, said ligand comprises residues 1-26.11 Binds to human TNF within the topological regions 7-128 and 141-153 do.

In a preferred embodiment of the invention, the ligand is human TNF (peptide 30 1) to a peptide having an amino acid sequence substantially identical to amino acids 1 to 18 of This is an antibody developed against.

In a preferred embodiment of the invention, said ligand is a monoclonal compound defined by MAb32. It is a local antibody. A sample of MAb32 produced by a hybridoma was obtained from a European N Collek/W N of Animal Cell Catchas (European C o11eeti.

n of Animal Ce1l Cultures+ECA CC)-' Vaccine Research and Product ion Laboratory), Noku Brick Health Laboratory Service Public Health Laboratory 5service ), Applied Micronoise Research Center (Centre for Ap plied Microbiology and Re5earch), Bo Tondown, Salisbury, Uist/Yar SF3 0JG, UK 1989 It was deposited on August 3, 2013, and was given acquisition number 89080302.

In a preferred embodiment of the invention, the method of treatment includes IL-2, AZT or Including co-administration of other anti-viral agents such as viruses.

The present invention provides a link between gamma interferon and the currently bound ligand TNF. It has also been found that there is a synergistic effect in the treatment of viral infections.

This synergistic effect is achieved through the use of endogenous TF and endogenous interferon. - Can be obtained either by administering TNF Liga/de alone. skilled in the art As is obvious to those who are Deaf: 1. Administration of anti-TNF ligand conjugated to TNF (endogenous interferon 2. anti-TNF ligand together with gamma interferon (endogenous 3.7NF); and anti-TNF ligand bound to 3.7NF Therefore, in a preferred embodiment of the method of treatment of the present invention used anti-TNF ligand alone or with TNF along with gamma interferon. including co-administration of either in combination.

A fourth aspect of the present invention is to use gamma interferon and anti-TNF ligand alone. or a combination thereof with TNF, wherein the ligand is When bound to F, the expression of endothelial coagulant activity of the TNF is inhibited, and the TNF Methods for treating viral infections in which the anti-viral activity of F. is unaffected or increased. The method consists of a composition used in the method.

In a preferred embodiment of the invention, when the ligand binds to TNF, TNF is Inhibited from binding to receptors on endothelial cells: tumor fibrin precipitates and TNF The tumor regression activity of TNF was increased; its cytotoxicity was not affected, and the tumor regression activity of TNF was increased; Further characterized in that tumor receptor binding activity is unaffected or increased. .

In a further preferred embodiment of the invention, said ligand has a topological geometry of residues 1 to 18. TNF epitopes defined by biological regions are naturally occurring biological Characterized by substantial inhibition of binding to active ligand.

In a still further preferred embodiment of the invention, the topological region of residues 1-30.1 17-128 and 141-135, more preferably the topological region of said residues 1-26. 117-128 and 141-153. Substantially inhibits pitope binding to naturally occurring biologically active ligands The ligand is bound to TNF in such a way that Such sequence regions are located in Figure 16. It is expressed topologically.

A fifth aspect of the present invention provides for the use of moths/meinterfeu/ and an anti-TNF ligand alone. or a combination thereof with TNF; A mammalian virus characterized by binding to human TNF residues 1 to 18. Compositions used to treat infections.

A sixth aspect of the present invention is to use gamma interferon and anti-TNF ligand alone. or a combination thereof with TNF, the ligand's force f, Within the topological region of residues 1-30, 117-128, and +41-153 A treatment for mammalian viral infections characterized by binding to human TNF It consists of a composition used.

In a preferred embodiment of this aspect of the invention, said ligand comprises residues 1-26.11 7-128, and 141-153. match. Such an array region is topologically represented in FIG.

In a preferred embodiment of the invention, the ligand is human TNF (peptide 30 A peptide having an amino acid sequence substantially identical to amino acids 1 to 18 of 1) ( This is an antibody raised against two.

In a preferred embodiment of the invention, said ligand is a monomer defined by MAb32. Al with clonal antibodies.

A seventh aspect of the invention provides anti-TNF ligands alone or in combination with TNF. production of drugs for the treatment of viral infections in mammals. In use, when the ligand binds to TNF, the endothelial coagulant properties of TNF expression of the activity is inhibited and the anti-viral activity of the TNF is not affected ( 1) It also consists of the use of an anti-TNF ligand characterized in that it is enhanced.

In a preferred embodiment of the invention, when TNF binds to a receptor on endothelial cells, Tumor regression due to tumor fibrils/precipitates and TNFI activity is increased, its cytotoxicity is unaffected, and tumor receptor binding of TNF is A further feature is that the synthesis activity is unaffected or increased.

In a further preferred embodiment of the invention, said ligand has a topology of residues 1 to 18. The epitopes of TNF defined by the scientific domain are naturally It is characterized in that it substantially inhibits binding to an agonistically active ligand.

In a still further preferred embodiment of the invention, the topological region of residues 1-30.1 17-128 and 141-135, more preferably the topological region of said residues 1-26, 117-128 and +41-153 Substantially inhibits pitope binding to naturally occurring biologically active ligands The ligand is bound to TNF in such a way that Such sequence regions are located in Figure 16. It is expressed topologically.

An eighth aspect of the invention provides an anti-TNF ligand alone or in combination with TNF. Use of either of the following: in the production of a drug for the treatment of viral infections in mammals; characterized in that the ligand binds to human TNF residues 1 to 18. The method consists of using an anti-TNF ligand.

Embodiments of system 9 of the present invention provide anti-TNF ligands alone or in combination with TNF. Use of either of the following: in the production of a drug for the treatment of viral infections in mammals; in which the ligand has residues 1-30, 11.7-128, and 141-15 Anti-TN characterized by binding to human TNF within a topological region of 3 Consists of the use of F riga/de.

In a preferred embodiment of this aspect of the invention, said ligand comprises residues 1-26.11 Binds to human TNF within the topological regions 7-128 and 141-153 do. Such an array region is topologically represented in FIG.

In a preferred embodiment of the invention, the ligand is human TNF (peptide 3° 1) to a peptide having an amino acid sequence substantially identical to amino acids 1 to 18 of This is an antibody developed against.

In a preferred embodiment of the invention, said ligand is a monomer defined by MAb32. It is a clonal antibody.

In a preferred embodiment for all aspects of the invention, said ligand is an antibody, F(ab) Fragments, reconstituted antibodies (CDR-branched humanized antibodies), nongludomains antibodies (dABs), single chain antibodies, anti-idiotype antibodies, serum binding proteins, selected from the group of receptors, and natural inhibitors. This ligand has a synthetic protein that is similar to one of the previous fragments or one of the previous fragments. and peptides. However, if the ligand is monoclonal or polyclonal, Preferably, the antibody is a null antibody or an F(ab) fragment thereof.

In this application, "tumor regression", "induction of endothelial coagulation", "induction of tumor fibrin adhesion", ”, “cytotoxicity”, and “receptor binding”. The biological activity of F is determined by the method shown below.

“Single domain antibody” as used in this application refers to Ward et al. (NatureS ] 1.544-546 (1989)). Used to indicate fragmentation.

Anti-idiotypic antibodies are further described in the following references: G, N, Galton and M,! , Gree 7, “Identification of biological receptors” "Otaib Pseudo", (Ann, Rev, lm5uno1., i, 253-2 80 (1986); K. Gita and P. A. Beaternon, “Cell surface Use of anti-idiotype antibodies as surface receptor probes ( Proc, Natl, Aead, Sci, U, S, A,, IJL, 2443-2447 (1978)).

In order to understand the essence of the present invention in more detail, the preferred deaf embodiment of the present invention will be described below. Examples and figures are included. In the figure: Figure 1 titrates MAb against TNF. The results of the analysis are shown below.

Figure 2 shows the anti-TNF monoclonal cytotoxicity in WEHI-164. Figure 3 shows the potency of antibodies 1 and 32.

Figure 3 shows the effects of anti-TNF MAb on the induction of endothelial cell coagulant activity by TNF. It shows the effectiveness of

FIG. 4 is a schematic illustration of epitopes on TNF.

Figure 5 shows the receptors of radioisotope-labeled TNF on aortic endothelial cells. shows the connection to the controller.

Figure 6 shows MAb32 (1), standard antibody (2), and Experiments on receptor binding of TNF forming a complex with and MAb47 (3) It shows.

Figure 7 shows MAb32 (1), standard antibody (2), and An experiment of receptor binding of TNF forming a complex with MAb47 (3) is shown. are doing.

Figure 8 shows MAb32 (1), standard antibody (2), and Experiments on receptor binding of TNF forming a complex with and MAb47 (3) It shows.

Figure 9 shows MAb32 (1), standard antibody (2), and We conducted experiments on receptor binding of TNF forming a complex with MAb47 (3). It shows.

Figure 1O shows MAb32 (1), standard antibody (2), and Experiments on receptor binding of TNF forming a complex with and MAb47 (3) It shows.

Figure 1112 MAb32 (1), Mark 1 antibody (2), and MAb47 (* ) k6, demonstrating efficacy against TNF-mediated abscess regression in vivo.

Figure 12 shows the monovalent Fab' flag 11MAb, MAb32, and MAb32. Fig. 3 shows the efficacy of anti-inflammatory drug therapy against TNF-mediated abscess regression in vivo.

Figure 13 shows standard MAb (1), MAb32 (2), and Vep+l'301 anti- Figure 3 shows the efficacy of serum (3) on TNF-induced abscess regression.

Figure 14 shows the reactivity of MAb 32 with overlapping peptides of 10AA length.

FIG. 15 shows a schematic three-dimensional diagram of the TNF molecule.

Figure 16 shows the topological diagram of the regions of residues 1-26, 117-128, and 141-153. It shows mathematically.

Figure 17 shows TNF alone and 200 μl of TNF-MAb32: (1) 2 μg of TNF; (2) 2 μg of TNF + Ab; (3) 4 μg of TNF; (4) Virus levels in ovaries after treatment with 4 μg of TNF + Ab: It shows.

Figure 18 shows TNF alone and 200 μl of TNF-MAb32; (1) 2 μg of TNF; (2) 2 μg of TNF + Ab; (3) 4 μg of TNF; (4) Shows the virus level in the lungs after administering 4 μg of TNF + Ab. Ru.

Figure 19 shows TNF alone and 200 μl of TNF-MAb32: (1) 2 μg of TNF; (2) 2 μg of TNF; (3) 4 μg of TNF; (4) Shows the virus level in the lungs after administering 4 μg of TNF + Ab; There is.

Figure 201! 4 days after infection TNF + 11500 Ab301; (1) PB S (N=9); (2) 2μgO) TNF + 1150 (1) Ab301 (N,,,7): (3) 6μg TNF + 11500 Ab301 (N=70 ); (4) 6 μg + 7) TNF + 11500 Ab301 (N = 4): (5) Treated with 8 μg of TNF + 1150 (1) Ab301 (N = 4): Figure 2 shows the titration of virus in the ovary of CBA/H7).

Figure 21 shows in vitro anti-inflammatory activity in L929 cells treated with TNF and MAb32. H3V-1 induction is shown.

Figure 2211Ab301; (1) TNF alone; (2) TNF +115 (1 24 hours after infection with various concentrations of TNF with or without Ab301: H3V-fig determination of ovaries of pre-treated mice is shown.

Is Figure 23 1? ! 11-7　N　F binding to L929 cells alone or with MA b32 or Ab301: (1) TNF alone; (2) TNF + MAb32; (3) TNF + A1 The case where 301: exists is shown.

L blood story l flea wind thought ball For all experiments, 10- to 12-week-old BAL obtained from the C3lRO animal facility. B/C female mice were used. Met (Math) Solid tumor and Met A ascites tumor The cell system is Sloan Kettering/Ganse/ Obtained from the laboratory of Dr. Lloyd J. Auld I. Tarr. WEHI-164 Fibrosarcoma The system is Australian National University, No. 7-Cathino Graduate School of Medical Sciences (John Cu. r! Gita of in 5chool of Medical Re5earch - Obtained from Dr. Dori.

Vibrator Everyone Dorma 4 Human recombinant pear TNFIO μg intraperitoneally of mice in complete Freund's anonymity gave immunity. One month later, TNF1 out of all 70 intra-adjacent bands. 0 μg was administered. After 6 weeks (4 days before fusion), the selected mice were given PBS ( Boost with μg of TNFIO in phosphate buffered solution . Radzien and Underwood (Mo1. Ismunol, 11.441 (1986)), lung cells from immunized mice were harvested from bone marrow. 5p210. Dilution on the supporting cell layer of mouse peritoneal macrophages secrete anti-TNF antibodies by radioimmunoassay by restricting We subcloned cell lines that were known to have the following properties. anti The body subclass is ELISA, Misotest, Commonwealth Serum Laboratory es)).

Ichijio Munoa Sei Iodination of TNF using Latokubaraoki/dase according to standard procedures did. Add 50μl of hybridoma culture supernatant to 12517NF (50μ). (20,000 cpm) at 4°C overnight in a thermostatic tube, -Se1) (cellulose coated with donkey anti-mouse/rat immunoglobulin, Wellcome Diagnostics )as))) was added and kept at room temperature (20°C) for an additional 20 minutes.

Following this incubation, 31 ml of PB was added and the tubes were centrifuged at 250 rpm for 5 minutes. separated. The supernatant was decanted and the pellets with bound radioactivity were counted.

Near body m gastropod Competitive assay using either immobilized antigen (LACT) or antibody (PACT) To determine the control specificity of monoclonal antibodies using standard methods, Aston and Eve Anil, Pharsac, Therapeu+, u, 403 (1985 )).

L five stand Flexible/pull microtiter tray (flexible 5icroti tre rrays) using a monoclonal antibody (sodium sulfate from mouse ascites fluid). Globulin precipitated with 100 μg/ml sodium bicarbonate buffer, 0.0 μg/ml sodium bicarbonate buffer. 05M, pH 9,6) and incubated overnight at 4°C to remove non-specific binding sites with PB. Blocked with 1% bovine serum albumin in S (BSA/PBS). various concentrations 12517NF immobilized antibody in the presence of a secondary anti-TNF monoclonal antibody of Decided to bind to. After adding antibodies and TNF at the same time and keeping the temperature constant for 24 hours, , wash 4 times with PBS, and count the wells (tells) with bound radioactivity. I got it. 100% binding was determined in the absence of xenogeneic monoclonal antibody and excess cognate 100% competition was determined in the presence of monoclonal antibodies. All dilutions are BSA /PBS was used.

L worker Purified with Broteino A and released in the presence of various concentrations of secondary monoclonal antibodies. TNF-coated microtamination of monoclonal antibodies prepared with radioisotope. binding to iter wells was determined. In the same way as above, use a microtiter plate. The plate was coated with TNF (50 Mg/m+). A large amount of competing antibody (50μ I) was previously kept at a constant temperature of 4°C on a plate for 4 hours, and then placed in a 125mm plate. Add noclonal antibody (30,000 cpm) and keep at constant temperature for another 24 hours. Ta.

After washing four times with PBS, binding to the wells was calculated. in the absence of competing antibodies 100% binding was determined and 100% competitive in the presence of excess unlabeled monoclonal antibody. The decision was made.

WEHI-164 Bioanalysis of recombinant TNF activity was performed by S.B.V., Ri and Ninoshino Meyer (J. lmmunol, Methods, 14.99 (1986)). Ta. By feeding monoclonal antibodies to cell cultures with ABT90, monoclonal The potency of null antibodies on TNF activity was determined.

1 Kanbooka fruitful TNF-induced tumor regression using monoclonal antibodies in the following three tumor abscess models. Bicutaneous abscess WEH evaluated for modulation of row activity! -164, MetA sarcoma, and ascites ToA abscess. Subcutaneous tumors were induced by injecting approximately 5×10 5 cells. This results in Approximately 14 days later, a tumor of 10-15 mm was formed. Human recombinant TNF (10Mg) and monoclonal antibody (200 μI ascites globulin) were administered to mice for 4 consecutive days. It was injected into the ascites. Stacked groups include PBS only or TNF and bovine growth hormone. A monoclonal antibody against Lumon was injected. At the beginning of each experiment, tumor size Caliper for solid tumors or the body of the tumor-bearing animal for ascites mice. Each was measured by weighing. These measurements were taken every day during the experiment. .

Ichiji - Receipt - Asei WEH1-164 cells that have grown to confluence are scraped off and added to Hang's balanced salt solution. Wash once with a solution containing 1% BSA in (HBSS, Gibco) did.

100 μl of unlabeled TNF (1-110,000 n/tube) or Noclonal antibody (ascites globulin from 1/10 to 1/10,000, 10 times Add 50 μl of 1251 TNF (50,000 cpm) Ta.

WEH+ cells (200 μl, containing 2×108 cells) were then added. child The mixture was kept constant for 3 hours in a stirred water bath at 37°C. End this constant temperature state At that point, 1 ml of HBSS was added and the cells were spun at 1600 rpm for 30 seconds. Ta. The supernatant was discarded and the bound 12517NF in the cell pellet was counted. .

All dilutions were made in HBSS containing 1% BSA.

of of of of 10% fetal calf serum (FCS), benin, streptomycin, and 2 - Conditioned at 37°C, 5% CO2 in RPMI-1804 containing mercabutoethanol. In this case, aortic endothelial cells (10 passages) were grown. Coagulant activity to TNF Therefore, in order to induce this, the experiment of Pevillat et al. (1986, PNAS, 83. 4533) and cultured cells in 24-well coaster trays. (Costar trays). Full fine monolayer with HBS After washing with S, TNF (0-500 units/medium) and monoclonal antibody ( diluted to m/200 with ascites globulin) was added. After 4 hours, scrape the cells. It was then frozen and sonicated.

Calcium reprecipitation time of normal donor platelet-deficient plasma at 37°C 100 μm cell lysate and 100 μm total cell coagulant activity. Cal chloride Add 100 μl of citrated platelet-deficient plasma to Cium (30 mM) to remove blood clots. The formation time was measured and recorded. TNF and/or monoclonal antibodies (final Experiment in which tumor cell culture supernatant was added to endothelial cells treated at a concentration of 1/2) I also went there.

12517<2L△2 in mice treated with TNF and monoclonal Yヱblood enters Effect of TNF and monoclonal antibodies on fibrin formation in vivo In order to investigate the (living body) was injected subcutaneously. After 7 to 14 days, when the abscess reaches a diameter of about 1 cm, TNF (10 Mg/body) and 125+ human fibrinogen (7,5μ g/living body, 122 μCi/mg 7 Amersham) alone or intraperitoneally in the presence of human TNF (200 μI/living ascites globulin). was injected into. As a standard monoclonal antibody, a monoclonal antibody against growth hormone A local antibody was used. Two hours after TNF fusion, the rut pieces were removed, weighed, and By counting the samples with a gamma counter, 1251 fibrillary/-gen mice were determined. The contamination of the tissue with water was confirmed. 13 monoclonal antibodies that reacted with human TNF All were separated. These monoclonal antibodies were MAt11MAbl1MAb1 2, MAb20, MAb21, MAb25. MAb31. MAb32, MAt +37, named MAb42, MAb47, MAb53, and MAb54 . Table 2 shows the efficacy of these monoclonal antibodies against the biological activity of human TNF. show.

As shown in Table 2, several monoclonal antibodies (MAbl, 47, and and 54) have the ability to inhibit both the promotion of coagulation and anti-tumor activity by human TNF ( Some antibodies that inhibit anti-tumor activity have been shown to inhibit t1ζ in vitro. No force 1 inhibits acceleration of coagulation (MAI) II, 12.25, and 53) . Indeed, MAb2+, which inhibits abscess regression, promotes coagulation in vivo.

Monoclonal fruit in TNF TNF biological activity 1111220212531! 2 3742475354 thin Cytotoxicity ---o--oo oo---- Tumor regression ---o--o+ oo-- -Induction of coagulability (endothelium) -00--00-0----Fibrin adhesion (abscess) ---++++++ O--Q-receptor binding (WEII+-164) -- -0--0+/HOO---O No effect - Inhibitory effect *For WEHI-164M tumor cells and tumor types, depending on MAb concentration (See Figures 3.13-17).

MAbs known to share epitopes on TNF from competitive binding studies 1, 47 and 54 are used to treat toxicity and other symptoms (where complete neutralization of TNF is required). Bacterial, viral, and parasitic infections with high TNF levels, such as ) have very favorable properties in the treatment of Other models like MAb32 Noclonal antibodies do not inhibit tumor regression, but they do inhibit the promotion of coagulation. It is more suitable as an adjuvant to be administered together with TNF during cancer treatment. This kind of treatment The form is used in cancer treatment where clotting may be promoted by TNF. cytotoxic drugs (e.g., biblastin, ancrouil, IFN alpha, IL- 2. Actinomy/inD, AZT, radiation therapy, atriamy/), myromy Non-C1/t/n, arabinonodo, donorton/,/sublatin, vinculi Sutin, 5-fluorouranol, pleomy/n (Watanahe Nu, 1-Peng uno pharmaeol, I+uunotoxico1. , Ill, 117-12 7 (1988>), or at some stage the TNF level was low (Narunaru Ship! ! (e.g. Maruba AI DS) and individual carcinomas derived from AIDS (e.g. Kaposi , non-Hodgkins lymphoma, and squamous) disease This is particularly necessary in conjunction with cytotoxic drugs.

MAt+32 (Figure 1) has an affinity for human TNF alpha that IgG2JK anti-I/L determined to be 8.77xl O-mo I/l based on It is the body. This monoclonal antibody is a human TNF beta (lymphotoquinone) and mouse TNF alpha.

As shown in Figure 2, according to the WEHI-164 assay, in vitro, MA b32 does not prevent TNF cytotoxicity.

Monoclonal antibody 32 inhibited BAL TNF induction of subcutaneously implanted WEHI-164 fibrosarcoma tumors in B/C mice. variably promotes the onset tumor regression activity (see Figure 11%). This feature is associated with TNF. Although not common to all monoclonal antibodies directed against larger receptors, of the binding portion of MAb32 such that the target mediates the uptake of TNF into abscess cells. The specificity is shown in Figure 4) (see Table 3).

Binding of 4% of TNF to WEHI-164's receipt at victim-watcher-ship 125 (-78F MAb dilution standard 11 MAB MAB 321/+0 141 1/lo0 8g 1/1.000 101 83 1/10.000 92 82 1/100.000 97 93 Enhancement of TNF activity by MAb 32 at lower doses of TNF was associated with similar levels of abscess To achieve the regression of The same thing happens (Fig. 1111P!).For the T-test at the level p<o, ot. 2.5 μg and 1 μg TNF in the 18th and 5 μg 1 in the 28th 2.5 μg and 1 μg are statistically significant. This degree of promotion also Administration of TNF is non-toxic and increases the survival rate of the subject. Figure 12 shows MAb3 The monovalent Fab fragment of 2, like the entire MAb 32, was also inhibited by TNF. has been shown to promote induced tumor regression (see below).

MAb32 is normally induced by incubating cultured cells with TNF. This inhibits the expression of coagulation factors on endothelial cells (see Figure 3). This response TNF receptors that are clearly different from the receptors found on cells but cannot be identified in advance. Mediated by scepter.

Conversely, MAb 32 has been shown to organize radiolabeled fibrinogen. sea urchin promotes in vivo activation of tumor bed coagulation . This is due to activation of mononuclear cell/macrophage coagulation and TNF -Gives further insight into the mechanism of induced tumor regression.

The results obtained with MAb 32 are compared with other anti-TNF MAbs in Table 2. show.

Regarding the potential of MAb32 and MAb47 to inhibit the binding of TNF to endothelial cells. I also tested it. Bovine aortic endothelial (BAE) cells (passage number 11) were grown in 24-well A culture dish (Corning) coated with gelatin (0.2%) in advance was used. (denatured) McCoy, implanted in >) and supplemented with 20% fetal calf serum. Grown to confluence in s5A medium. Radioactive receptor assay (cold All dilutions (of TNF and MAbs) were performed in this medium. The BAE cells are , cold TNF (O to 10100n, or MAb (1/100 to 1/ ascites globulin diluted to 100,000) and iodinated TNF (500,000 00 cpm) for 1 hour. At the end of this time, remove the medium Cells were washed with 1M sodium hydroxide prior to lysis. the cell msl fluid Bound radioactive TNF was counted. Specific binding of labeled TNF to cells is determined in this way.

MAb32, MA147 and I! Results obtained with this assay using IIMAb is shown in Figure 5.

Coagulation assay using BAE cells cultured in the presence of TNF and anti-TNF (MA+) The results obtained are related to those obtained with the BAE radioreceptor assay. .

That is, it inhibits the expression of coagulation factors on the surface of endothelial cells and reduces the coagulation time compared to TNF alone. ), as well as its receptors for TNF. It also inhibits the binding of This is exemplified by MAb32 and MAb47.

MAb32, which does not inhibit TNF binding to WEHI-164 cells, inhibits TNF internalization. Binding to skin cells is inhibited. This result suggests that another functional site exists in the TNF molecule, These functional sites may be linked to distinct receptor subpopulations in different cell types. This supports the hypothesis that there is an interaction with ulation 3). Therefore Ligands that bind to defined regions of TNF are Biological effects of TNF by limiting its binding to subtypes It is possible to correct.

As shown in Figure 5, MAb47 has a particularly strong effect on the interaction of TNF with endothelial cells. It is an inhibitor. Noy of specific binding at dilutions from 1/100 to I/10000 The cent is essentially zero.

1. In vitro treatment of 15 M2" or human F. Δ It has already been mentioned that MAb 32 enhances the anti-tumor activity of human TNF. Beta. The mechanism behind its enhancement is that TNF binding to specific (W tumor) receptors. Limited to subspecies and not bound to other species (endothelial), and Subsequently, it is thought that the aim is to reduce the toxicity of TNF to non-tumor cells. . In this mechanism, TNF uptake by tumor cells has been shown in in vitro studies. There is no need to reinforce the structure. Additionally, MAb32 has been shown to be effective in certain human carcinoma cell lines. Facilitates direct binding of human TNF to certain TNF receptors.

1st ward stands For the following human carcinoma cell lines, the presence of MAb32 enhanced Additionally, receptor-mediated uptake of TNF was assayed. The human carcinoma cell line is BIOlCaCo, HT29.5KCOI (all of the above are colon carcinomas), 56 37 (bladder carcinoma), MM418E (melanoma), IGR3 (melanoma), MCF7 (breast carcinoma). These cells were incubated with 10% fetal serum, Beni/Lin/Su RPMI-1640 (MM4) with Trebutomain and L-glutamine 18E) DMEM (CaCo and IGR3) or eye scope (lscov es) Modified DMEM (BIO1HT29.5KOI, 5637, MCF7) +7) grown in either. Receptor assays are performed on endothelial cells as described above. It was done for cells.

However, for iodinated TNF, all incubation times were extended to 3 hours, and the radiolabel Only the recognized BIO cells were cultured for 1 hour.

The presence of MA b32 inhibits the melanoma cell lines MM418E and I tested. GR3 (shown in Figure 13.14), Bladder Cancer 115637 (shown in Figure 8), and Breast Enhanced TNF uptake by carcinoma MCF7 (shown in Figure 9) was observed.

MAb32 can be used in any other cell line, as shown for B10 (shown in Figure 1O). There was also no effect on TNF-receptor interactions in the strains. MAb 47, blocking TNF binding to WEHI-164 cells and endothelial cells. and also inhibited TNF-mediated tumor regression, which was shown to inhibit all It was found that TNF binding was significantly inhibited in cell line I (Figures 6-10). .

1 residue Receptor binding analysis reveals a second mechanism by which MAb32 promotes the anti-tumor activity of TNF. It shows the structure. This second pathway to TNF enhancement is due to the presence of MAb32. This is the result of increased uptake of TNF by all receptors in the tumor.

In vitro treatment with MAb32 or the -FAb' fragment of MAb32 TN -'-'s Mice bearing WEHI-164 subcutaneous tumors (N-5 animals/group) as described above. An intestinal tumor regression test was conducted on the following. Abscess size was measured daily throughout the study period. .

The results obtained using MAb32 are shown in FIG. This result will be displayed when processing is completed (2 (MAb32: [3 days). Quasi-MAb: Kai MAb47). Treatment of standard MAb-TNF and MAb32-TNF Differences observed between groups are statistically significant by T-test at the pro, 01 level. There is.

The results using the monovalent FAb' fragment of MAb32 are shown in FIG. of tumor Size was measured daily throughout the test period. This result will be displayed upon completion of processing (2nd day). The mean +/-3D% change in tumor area is shown. Standard - TNF and MAb32 - observed during the treatment group with TNF! ! The growth is T- at the p<0.01 level. The test was statistically significant.

′−: −peb de Figure 13 shows TNF plus standard MAb (Latin) against peptide 301 over 3 days. anti-growth hormone), TNF+MAb32 or TNF+ antiserum (antibody against growth hormone) Changes in the tumor area in abscess-bearing mice treated with Robulin fraction It shows. In the unpaired test, the standard group was the test group (MAb32, Antiserum 301) and both antiserum 301) were clearly observed. On the other hand, MAb32 and peptide anti-blood There is almost no difference between it and Sei 301. (The standard vs. MAb32 is p 0.002; p<o, 025 for standard versus peptide 301). In this way, M Antisera obtained using peptides that constitute part of the specificity of Ab32 also NF enhances ffl tumor regression.

As seen in Figure 4, the competitive binding assay! 3 monoclonal antibodies are divided into two major types. showed that it can be done. i.e. MAb+, 21.47.54.37.32 and 25 and MAbll, 12.53, and 42. Next, this To identify the region on human TNF confirmed by the monoclonal antibody of A test was conducted.

Five mechanisms of human TNF confirmed by monoclonal 1. Geison et al.'s method (Geison et al., PNAS, 11.3998-4002 (1984>) li: 7 and 10 amino acids on polypropylene bottles. Peptides with overlapping residue lengths were synthesized. This overlap is Consisting of 6 and 9 residues, respectively, these peptides are precisely related to TNF. Completes the entire amino acid sequence. For these peptides, ELISA Therefore, the reactivity with MAb was examined. At the same time, pre-incubation with whole TNF MAbs with TNF activity absorbed from them are also tested using an inert standard. The peptide activity was tested as follows.

2. A longer chain peptide of TNF was synthesized as follows. These pages Putide was used to increase the antiserum properties of sheep using the following standard method. It is. Treating 797 sheep species with TNF peptide coupled with ovalbumin, Emulsified in Complete Freund's Aginiband and treated with peptide-opalbumino and serum for 4 weeks. The cells were incubated for 1 hour and analyzed for the presence of anti-TNF antibodies by Radioimmunoa.

Among these peptides, peptides 275.301.305.306 and 307 Only serum that reacted with total TNF was elicited. These active sera were then A competitive binding test (PACT test method) was conducted with MAIl.

The following peptides were synthesized. These peptides are commonly used for each amino acid. The TNF sequence region is shown in parentheses.

Nie two two! 21 H-Al a-Lys-Pro-Trp-Tyr-Gl u-Pro-+ 1 e-Tyr-Leu-OH (111-120) Twenty two! Up H-Vat-Arg-3er-8er-Ser-Arg-Thr-Pro-3e r-Asp-Lys-Pro-Val-Ala-His-Val-Val-Al a-OH(1-18) Nienini 1Ll H-Leu-Arg-Asp-Asn-Gln-Leu-Vat-Val-Pr o-5er-Glu-Gly-Leu-Tyr-Leu-11e-OH(43- Nib 1JLLLL H-Leu-Phe-Lys-Gly-Gin-Gly-Cys-Pro-3e r-Thr-His-Val-Leu-Leu-Thr-Hls-Thr-11 e-9er-Arg-11e-OH (63-83) Nicosey L11”- H-Leu-Ser-Al　a-Glu-11e-Asn-Arg-Pro-A sp-Tyr-Leu-Asp-Phe-Ala-Glu-9er-Gly-G ln-Val-OH(132-150) peptide 306 ! (-Va l-, Al a-Hi 5-Va 1-Val-Al a-Asn -Pro-Gln-Ala-Glu-Gly-Gln-Leu-OH (13-2 6) SLLL H-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-As n-Arg-Arg-Ala-Asn-Ala-Leu-Leu-Al　a-A sn-GI Y-OH (22-40) peptide 308 H-GIY-Leu-Tyr-Leu-11e-Tyr-5er-Gln-Va l-Leu-Phe-1,ys-Gly-Gln-Gly-OH (54-68) peptide 309 H-His-Val-Leu-Leu-Thr-His-Thr-11e-3e r-Arg-11e-Al a-Va l-5er-Thr-Gln-Thr- Lys-Va 1-Asn-Leu-Leu-COOH (73-94) nib l" 2m H-Thr-11e-Ser-Arg-11e-Ala-Val-Ser-Th r-Gl n-Thr -OH (79-89) These peptides are commonly It was synthesized using a standard method.

All peptides were synthesized using the Fmo c-polyamide method, which is a solid-phase peptide synthesis method. Cert/Ra, J, Chat Soc, Chet Co+nun, 1 1.537-539 (1978)). The solid resin used was Pep Syn KA, this is on a KieseLguhr support. 4-hydroxymethyl as a functional group linking agent in polydimethylacrylamide gel. It supports fenoquine acetic acid (Atherton et al., Am, Chek. 5ac1. u, 65g4-6585 (1975)).

The carboquine terminal group amino acid is a DCC/DMAP-mediated symmetric anhydride ester. It was immobilized on a solid support by the silica method.

All Fmoc clusters were washed away with piperidine/DMF and then peptide conjugated. , except for some amino acids, via pentafluorophenyl active ester or Or BOP/NMM/HOBt (Castro reagent) (Fournier et al.

Int, J, Peptide Protein Red, il, +33 -139 (1983)).

The method is shown in Table 4.

Side chain protecting groups of amino acids were removed simultaneously with dissociation. However, Ac on / Stemono m was left until after synthesis.

Table 4 L engineering L1 (iLILil　　　〱fiArg　Mtr or Pmc　Either Asp　0But　Either Cys Arnc (permanent) Either Glu 0 But His BoC0Pfp method only Lys BoCL) Either one? Ser But BOP method only Thr But BOP method only Tyr　But　either Trp None or either Asn None OF’fp method only Gln None QPfp method only Carp JLJLIL Peptide 301.302.305 was prepared using 95% TFA and 5% thioanisole. 1.5 hours), reversed phase C4 column (buffer A-0,1% TF) A aqueous solution, purified on Babfa 8-80% ACN 20% A).

Peptide 303.304 was cleaved from the resin using 95% TFA and 5% phenol. (separated for 5-6 hours) and purified on a reversed phase C4 column (buff 7a same as above). . Peptide 306.308 was dissociated from the resin using 95% TFA and 5% water. (1.5 hours) and purified on a reverse phase C4 column (Bubfluors were the same as above).

Peptide 309 was dissociated from the resin using 95% TFA and 5% thioanisole. (1.5 hours) and purified on a reverse phase C4 column (buffer yll same as above).

Peptide 307 was prepared using 93% TFA, 3.1% anisole, 2.97% ethyl methane. Dissociated from resin using a mixture of lusulfide and 0.95% ethanedithiol (3 hours) and purified on a reverse phase C4 column (buffers were the same as above).

1st coming Typical results of MAb ELISA using 7 and 10 conjugates are shown in Figure 21.

In conjunction with the results of the PACT test using sheep anti-peptide serum (shown in Table 6) Thus, the following regions of TNF contain binding sites for anti-TNF MAbs:

MAbl: Residues 1-18.5g-65, 115-125, 138-149 MAb ll: residues 49-98 MAbl2: residues 22-40.70-87 MAb21: residues 1-18.76-9 076-9O: Residue 12-22.36-45.96-105.132-157M Ab32: residue 1-26.117-118.141-153 MAb37: residue 2 2-31.146-157MAb42: residue 22-40.49-96.110- 127.136-153MAb47: Residues 1-18.108-1108-l28 : residue 22-40.69-97.105-128.135-155 MAb54: Residues 56-79.110-127.136-155 Table 5 Competitive binding of TNF by anti-TNF monoclones in the presence of anti-peptide serum MAb/peptide serum Note 1 Nee indicates no competition, + indicates high concentration (t/SO) of anti-peptide antiserum. ++++ indicates slight competition for anti-peptide serum equivalent to that of the homologous MAb. indicates strong competition. Note 2 = This test draws serum to confirm total TNF. Only subebutide was used.

1 difference”””- Peptide 301 combined with ovalbumin in the 70inzuAji 1 band was found in sheep. made immune. Antiserum reactive with peptide 301 was obtained from this immunized sheep. Ta.

class n The arrangement of regions identified by each MAb indicates the following. That is, Guru The MAb (MAbl, 21.47.54.37.32 and 25) of group As shown in the figure, except for MA b 37 and 54, the region of residues 1-18 is and binds to TNF. On the other hand, MAt+ (MAb I1゜12.53 and H42) It, so-called baren on the three-dimensional structure of TNF It binds to TNF in the region of residues 70-96 surrounding the dromic ring. endothelial thin MAbs (MAbl, 32.42.47.54 and and 53), which are also three-dimensional models with residues 108-128 containing a cyclic structure. Combine within a region. This suggests that this region is found in endothelial cells rather than tumor cells. This is thought to indicate that the protein interacts with the TNF receptor. in vivo MAb 32, which promotes tumor regression and antiviral activity of TNF, has residues 1-26. 117-128, and all the annular regions corresponding to +41-153. therefore, binding to these regions enhances the biological activity of TNF and At the same time, it is a decisive factor in reducing toxicity to normal cells.

As is clear from Table 2, MAbl, 47 and 54 are identical to the biological activity of TNF. brings about the effect of From the above results, these three monoclonals are mediated by TNF molecules. join to the same area. Therefore, mainly the region of residues 1-20, residues 56-77 A group consisting of the region of , the region of residues 108-128, and the region of residues 138-149 A ligand that binds TNF in at least 62 regions selected from the loop is M It appears to contribute to the biological activity of TNF in a similar manner to Abl, 47 and 54. Ru. Similarly, TNF binds primarily in the region of residues 1-20 and 76-90. We believe that Surigant has a similar effect to MAb21 on the biological activity of TNF. It will be done. Binds primarily to TNF in the region of residues 22-40 and 69-97. would have a similar effect on the biological activity of TNF as MAbl2. . Mainly in the region of residues 1-30, 117-128, and 141-153 Ligatsu, which binds to TNF, has a similar effect to MAb 32 on the biological activity of TNF. It is expected that Also, mainly residues 22-40.49-97.110 The ligand that binds to TNF in the -12f and +36-153 regions is It is expected to have a similar effect to MAb42 on the biological activity of F. mainly The ligand that binds TNF in the region of residues 22-31 and 146-157 is , is expected to have a similar effect to MAb37 on the biological activity of TNF.

Mainly residues 22-40, 69-97, 105-128 and 135-15flN 7) Ligands that bind TNF in the region have a significant effect on the biological activity of TNF. It is expected that it will have similar effects.

The present inventors have demonstrated that the biological activity of TNF is altered by the binding of a ligand to TNF. The effect on biological activity is related to the specificity of the ligand. This was shown very clearly. For example, residues 1-26.117-128, and Binding of MAb32 to TNF in the region 141-153 is associated with endothelial coagulation of TNF. The result is induction of solid activity and blocking of TNF binding to receptors on endothelial cells. induces tumor fibrin precipitation and enhances the tumor regression activity of TNF, and , does not affect cytotoxicity, but does affect tumor receptor binding activity of TNF. Do not detract or increase. This effect on the biological activity of TNF may be due to MAb3 Naturally occurring biological activity of epitopes of TNF identified by 2 believed to originate from interference with binding to the Therefore, by MA b 32 Similar effects may also be produced if the epitope identified by MAb32 naturally A similar mechanism is used to prevent binding to biologically active ligands. It is believed that this is caused by the ligand binding to the region of TNF. This conclusion Interference with binding may be due to steric hindrance or other mechanisms.

Therefore, the MAb 32 described here against a naturally occurring biologically active ligand Prevention of binding of epitopes identified by Ab301 and Ab301 is also within the scope of the present invention. Please emphasize that (.

-NF b's TNF was added to the human Alf virus in vitro. (Mestan et al., Nature, 'JjJ, 816-819 (1986), Wang and Godel, Nature, Mu 1.819-H2 (1986)) , and in vivo (Doherty et al.

J, 1jnuno1. , Eng., 376-3110 (1989)). It is known that it exhibits antiviral effects. The present inventors discovered that TNF- Effect of MAb32 complex on the anti-viral effect of TNF in mice infected with vaccinia. I experimented with sound.

CBA-H mice were treated with vaccinia (107 pf u VV-HA-TK, Lamb 71- and others.

24 hours before infecting Nature, ljJ, 44-46 (107)). , the mice were treated with TNF alone (reconstituted human TNF), or mixed for 24 min before inoculation. The cells were treated with TNF and MAt) 32 (200 μm ascites globulin). homogenize , ovaries, lungs, and lung samples treated with trypsin (1 mg/ml) after 4 days. 143B indicator cell line (indicator cell 1i ne) was used to determine the virus titer.

Mice treated with TNF-MAb32 compared to mice treated with TNF alone. As a result, virus levels decreased in the ovaries (Figure 17), lungs (Figure 18), and lungs (Figure 19). You can see that

Similar results were obtained in mice treated with TNF and antibody 301 (Figure 20).

In vivo, 1 virus Hv-) CBA/H mice were infected with 107 pfu of monovirus. 24 hours before infection with pure herpes virus 1 (H8V-1), appropriate TNF Treated with +/- Ab301 administration. After diluting the antibody to 1150, 6.0 mic of TNF was mixed and the mixture was left at room temperature for 1 hour. This TNF+ TNF complexed with various concentrations of At+ was extracted from the stock of Ab301, Dilute in PBS (i.e. 0.5-2.0 micrograms). That mouse is After 3 days of infection, the animals were sacrificed and the ovaries were removed aseptically. these Organs were homogenized in PBS and treated with 100 μl O, 1% Tribunone for 30 min. did. Trib//treatment was terminated by adding Fe2. this sample were serially diluted in 1/10 increments. These diluted solutions were incubated for 1 hour at 37°C. Absorb into cell (Vero cell) and top coat with F15 + 5% FC9 for 2 days. It was left at 37°C for a while. Spots were counted and virus concentration calculated. The results are shown in Figure 22. show.

In vitro ni゛(v- L929 cells (Linbro) 24 w e l! plate ) At a concentration of 500,000 cells/well, TNF alone (10-4 oong) or TNF and MAI) 32 (complex method as described above).

These cultures were left for 24 hours and then incubated with HSV-1(0,1-2,0). Infected. 1 hour on the cells to absorb 100 μl of virus-containing PBS. Leave it at 37℃ for a while, remove excess virus, and top coat with F15 + 5% FC3. Ta. These cultures were left for 48 hours.

After that time, freeze and thaw the cells twice, and drain the supernatant continuously. diluted (trypsinization not required). Then, as mentioned above, absorb on the vero cell. harvested and grew. The results are shown in FIG.

125 - of (b 2 Different dilutions of MAb32 or At)301 in RPMI + 10% FCS Then, 2.0×10B L929 cells, also in RPMI, were subsequently iodinated. TNF (50,000 cpm, 150 μl) was added. These processed materials at 3 o'clock After culturing for a period of time, the cells were washed once and pellets were counted. vinegar All treatments were performed three times. The results are shown in FIG.

- TNF Gant - Gamma mine that is based on the TNF virus - Feron Bon left Windset Star [! Ab3o1 complexed with TNF was diluted with 6μgO after diluting Ab301 to 11501M. ) was prepared by mixing TNF. The TNF-10 antibody was administered. Also, 105υ/mouse of murine gamma inotaferon (mlFN-γ) alone administered. TNF was also administered with the Ab301+m1FN-γ treatment. m 1ps-7 until after 1 hour room temperature incubation of TNF and antibody. ? They were not mixed into one mass.

Twenty-four hours later, the mice were injected intravenously with 108 pfu of smallpox. Results of this experiment are shown in Table 6.

Cytokine treatment LogIO virus titration (pfu/ml) ±EM PBS (n=5) 8.20±0.296μg TNF+1150 Ab301 5.93±0.33105U m1FN-7 (n=5) 5.66±0.35 6μg TNF+1150 Ab301 <2.0+105U m1FN-7 As can be seen from the results shown in Table 6, m1FN-7 and MAI) 301: TNF There is a remarkable synergistic anti-viral effect with the complex.

As already seen in Figures 17.18 and 19, the M Administration of TNF in combination with Ab32 reduces the amount of virus that can be recovered from infected animals. resulting in a reduction in the number of particles. Although not supported by scientific theory, anti-TNF Enhanced anti-viral effects produced by administration of TNF in combination with ligand The rus effect may be caused by interference with the binding of TNF to receptors in the endothelium or by infection with a virus. TNF seen in a direct increase in TNF binding to cellular receptors (Figure 23) This is believed to be the result of increased amounts of ligand. Therefore, the method of the present invention It can be particularly applied to the treatment of viral infections, not limited to infections on the skin surface. I can believe it. In particular, the method of the invention is applicable to the treatment of infections caused by the following viruses: In other words, hepatitis, AIDS, herpes, viral meningitis, and green disease. (Grsen 5onkey) virus and smallpox eruption.

It will be appreciated by those skilled in the art that the invention described above departs from the generally known spirit and perspective of the invention. It will be appreciated that many changes and modifications can be made without .

Figure 5 monoclonal antibody (IoglOng) Figure 6 monoclonal antibody (loglo ng) Figure 7 Figure 8 monoclonal antibody (loglOng) Figure 9 monoclonal antibody (loglo ng) Figure 10 XJII Gate Ari Figure 12 process (200 μi globulin + 10 μg TNF injection/day/mouse) Figure 13 =Rumor C・Cheer■°Den o4 Figure 15 Figure 16 logl O virus titration value (pfu/ml) LogIO virus titration value (pf u/ml) Log I O virus titer value (pfu/ml) Continued from front page Country of Australia 2611 Australia

Claims (1)

[Claims] 1. administering an anti-TNF ligand to a mammal, either alone or in combination with TNF. In a method for treating viral infection in mammals, the method comprises administering When the compound binds to TNF, the expression of the endothelial coagulant activity of the TNF is inhibited. and that the anti-viral activity of the TNF is unaffected or enhanced. Characteristic methods for treating viral infections. 2. When the ligand binds to TNF, the TNF receptor on the endothelial cell tumor fibrin precipitation and TNF-induced tumor regression activity are enhanced. its cytotoxicity is not affected and the tumor receptor binding activity of TNF is not affected; Viral activity according to claim 1, further characterized in that the virus is not affected or is enhanced. How to treat infections. 3. the ligand is defined by a topological region of residues 1 to 18; TNF epitopes bind to naturally occurring biologically active ligands. The treatment for viral infection according to claim 1 or 2, characterized in that the method substantially inhibits Treatment method. 4. topological regions of residues 1-30, 117-128, and 141-135, More preferably, topological regions 1-26, 117-128, and The epitopes of TNF defined by T The method for treating viral infection according to claim 1 or 2, wherein the method is made to bind to NF. 5. administering an anti-TNF ligand to a mammal, either alone or in combination with TNF. In a method of treating a viral infection in a mammal, the method comprises administering A viral virus characterized by binding to residues 1 to 18 of human TNF. How to treat infections. 6. administering an anti-TNF ligand to a mammal, either alone or in combination with TNF. In a method of treating a viral infection in a mammal, the method comprises administering the topological regions of residues 1-30, 117-128, and 141-153. A method for treating a viral infection characterized by binding to human TNF within a range. 7. The ligand has a phase of residues 1-26, 117-128 and 141-153. The method according to claim 6, wherein the method binds to human TNF within a geometric region. How to treat viral infections. 8. The ligand comprises amino acids 1 to 18 of human TNF (peptide 301). An antibody raised against a peptide with a substantially identical amino acid sequence. The method for treating viral infection according to claim 3 or 5, characterized in that: 9. The ligand is a monoclonal antibody defined with MAb32. The method for treating viral infection according to any one of claims 1 to 7, characterized in that: 10. The method of treatment includes other anti-inflammatory agents such as IL-2, AZT or acyclouyl. The method according to any one of claims 1 to 9, characterized in that it involves co-administration of a viral drug. How to treat viral infections. 11. The treatment method includes gamma interferon and anti-TNF ligand alone. or in combination with TNF. 11. The method for treating a viral infection according to any one of 1 to 10. 12. Gamma interferon and anti-TNF ligand alone or together with TN F and either one of the combinations, and when the ligand binds to TNF, When the expression of the endothelial coagulant activity of the TNF is inhibited, and the anti-viral activity of the TNF is inhibited. treatment of viral infections characterized by unaffected or enhanced viral activity; Compositions used for medical treatment. 13. When the ligand binds to TNF, the TNF receptor on endothelial cells is activated. tumor fibrin precipitation and TNF-induced tumor regression activity are increased. its cytotoxicity is unaffected and the tumor receptor activity of TNF is Composition according to claim 12, further characterized in that it is unaffected or enhanced. . 14. the ligand is defined by a topological region of residues 1 to 18; TNF epitopes bound to naturally occurring biologically active ligands. 14. The composition according to claim 12 or 13, wherein the composition substantially inhibits. 15. Topological regions of residues 1-30, 117-128, and 141-135 , more preferably, the topological region 1-26, 117-128 of the residue, and 141-153, the epitope of TNF is naturally occurring. the biologically active ligand in such a way that it substantially inhibits binding to the biologically active ligand. 14. The composition according to claim 12 or 13, which binds to TNF. 16. Gamma interferon and anti-TNF ligand alone or together with TN F, and the ligand is a human TNF residue 1 used in the treatment of viral infections in mammals characterized by binding to 18 from composition. 17. Gamma interferon and anti-TNF ligand alone or together with TN F and the ligand comprises residues 1-30, 117- 128, and binds to human TNF within the topological region 141-153. A composition for use in treating viral infections in mammals, characterized in that: 18. The ligand is located at residues 1-26, 117-128 and 141-153. Claim 15 or Claim 15, characterized in that it binds to human TNF within a topological region. is the composition described in 17. 19. The ligand comprises amino acids 1 to 18 of human TNF (peptide 301). is an antibody raised against a peptide that has an amino acid sequence substantially identical to The composition according to claim 15 or 17, characterized in that: 20. The ligand is a monoclonal antibody defined by MAb32. The composition according to any one of claims 11 to 19, characterized in that: 21. anti-TNF ligand, either alone or in combination with TNF, in mammals. For use in the production of a medicament for the treatment of viral infections in animals, the ligand is When binding to TNF, the expression of endothelial coagulant activity of the TNF is inhibited, and characterized in that the antiviral activity of the TNF is unaffected or enhanced. Use of anti-TNF ligands. 22. When the ligand binds to TNF, the TNF receptor on endothelial cells is activated. Waiting for the tumor is inhibited: tumor fibrin precipitation and tumor regression activity by TNF is increased. advanced: its cytotoxicity is not affected and the tumor receptor binding activity of TNF is Use according to claim 21, further characterized in that it is unaffected or enhanced. 23. the ligand is defined by a topological region of residues 1 to 18; TNF epitopes bound to naturally occurring biologically active ligands. 23. The use according to claim 21 or 22, characterized in that it substantially destroys. 24. Topological regions of residues 1-30, 117-128, and 141-135 , more preferably, the topological region 1-26, 117-128 of the residue, and 141-153, the epitope of TNF is naturally occurring. the biologically active ligand in such a way that it substantially inhibits binding to the biologically active ligand. 23. The use according to claim 21 or 22, wherein the use is coupled to TNF. 25. anti-TNF ligand, either alone or in combination with TNF, in mammals. For use in the production of a medicament for the treatment of viral infections in animals, the ligand is Anti-TNF ligand characterized by binding to residues 1 to 18 of human TNF Use of. 26. anti-TNF ligand, either alone or in combination with TNF, in mammals. For use in the production of a medicament for the treatment of viral infections in animals, the ligand is Within the topological region of residues 1-30, 117-128, and 141-153 Use of an anti-TNF ligand characterized in that it binds to human TNF. 27. The ligand is located at residues 1-26, 117-128 and 141-153. 27. Binds to human TNF within a topological region. Use of. 28. The ligand comprises amino acids 1 to 18 of human TNF (peptide 301). is an antibody raised against a peptide that has an amino acid sequence substantially identical to 26. Use according to claim 25, characterized in that: 29. The ligand is a monoclonal antibody defined by MAb32. 29. Use according to any of claims 21 to 28, characterized in that: 30, the above-mentioned ligand may be used for antibodies, F(ab) fragments, reconstituted antibodies (CD R-branched humanized antibodies), single domain antibodies (dABs), single chain antibodies, anti-I Among the group of diotypic antibodies, serum binding proteins, receptors, and natural inhibitors 10. A method according to any one of claims 1 to 9, characterized in that the method is selected from: 31. The ligand is a monoclonal or polyclonal antibody, or 31. The method according to claim 30, wherein the F(ab) fragment of