EP0608212A1 - Method of treating viral infection - Google Patents

Method of treating viral infection

Info

Publication number
EP0608212A1
EP0608212A1 EP91915297A EP91915297A EP0608212A1 EP 0608212 A1 EP0608212 A1 EP 0608212A1 EP 91915297 A EP91915297 A EP 91915297A EP 91915297 A EP91915297 A EP 91915297A EP 0608212 A1 EP0608212 A1 EP 0608212A1
Authority
EP
European Patent Office
Prior art keywords
tnf
ligand
residues
binds
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP91915297A
Other languages
German (de)
French (fr)
Other versions
EP0608212A4 (en
Inventor
Deborah Ann Rathjen
Roger Aston
Ian Alastair Ramshaw
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Teva Pharmaceuticals Australia Pty Ltd
Original Assignee
Peptide Technology Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peptide Technology Ltd filed Critical Peptide Technology Ltd
Publication of EP0608212A4 publication Critical patent/EP0608212A4/en
Publication of EP0608212A1 publication Critical patent/EP0608212A1/en
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241Tumor Necrosis Factors
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K39/395Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/525Tumour necrosis factor [TNF]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with tumour necrosis factor alpha (TNF) .
  • the ligand is characterised in that the binding of the ligand to the TNF is such that the biological activity of TNF is modified.
  • the invention also relates to a composition for treating viral infection.
  • Tumor necrosis factor alpha is a product of activated macrophages first observed in the serum of experimental animals presensitized with Bacillus Calmette-Guerin or Corynebacterium parvum and challenged with endotoxin (LPS) . Following the systematic administration of TNF hae orrhagic necrosis was observed in some transplantable tumours of mice while in vitro TNF caused cytolytic or cytostatic effects on tumour cell lines. In addition to its host-protective effect, TNF has been implicated as the causative agent of pathological changes in septicemia, cachexia and cerebral malaria. Passive immunization of mice with a polyclonal rabbit serum against TNF has been shown to protect mice against the lethal effects of LPS endotoxin, the initiating agent of toxic shock, when administered prior to infection.
  • TNF has been cloned allowing the usefulness of this monokine as a potential cancer therapy agent to be assessed. While TNF infusion into cancer patients in stage 1 clinical trials has resulted in tumour regression, side-effects such as thro bocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension have also been reported. These quite significant side-effects associated with the clinical use of TNF are predictable in view of the many known effects of TNF, some of which are listed in Table 1. TABLE 1 BIOLOGICAL ACTIVITIES OF TNF -ANTI-T ⁇ MOUR -ANTI-VIRAL -ANTI-PARASITE
  • tumour cells pyrogenie activity angiogenic activity inhibition of lipoprotein lipase activation of neutrophils osteoclast activation induction of endothelial, monocyte and tumour cell procoagulant activity induction of surface antigens on endothelial cells induction of IL-6 induction of c-myc and c-fos induction of EGF receptor induction of IL-1 induction of TNF synthesis induction of GM-CSF synthesis increased prostaglandin and collagenase synthesis induction of acute phase protein C3
  • TNF activation which occurs as a consequence of TNF activation of endothelium and also peripheral blood monocytes.
  • Disseminated intravascular coagulation is associated with toxic shock and many cancers including gastro-intestinal cancer, cancer of the pancreas, prostate, lung, breast and ovary, melanoma, acute leukaemia, myeloma, myeloproliferative syndrome and myeloblastic leukaemia.
  • Clearly modifications of TNF activity such that tumour regression activity remains intact but other undesirable effects such as activation of c agulation are removed or masked would lead to a more advantageous cancer therapy. Complete abrogation of TNF activity is sought for successful treatment of toxic shock.
  • the present inventors have produced panels of monoclonal antibodies active against human TNF and have characterised them with respect to their effects on the anti-tumour effect of TNF (both in vitro and in vivo) , TNF receptor binding, activation of coagulation (both in vitro and in vivo) and defined their topographic specificities.
  • This approach has led the inventors to show that different topographic regions of TNF alpha are associated with different activities.
  • This work is described in detail in a co-pending patent application filed under the Patent Cooperation Treaty in the Australian Receiving Office on 7 August 1990 and disclosure of this application is incorporated herein by reference.
  • the present inventors have made the surprising finding that the administration of TNF in combination with a specific anti-TNF ligand provides an effective anti-viral treatment.
  • the administration of the specific anti-TNF ligand alone will provide an effective anti-viral therapy as the ligand will bind to endogenous TNF, thereby providing the same effect as if the anti-TNF ligand was administered in combination with TNF.
  • This administration of the anti-TNF ligand alone may be the preferred method of therapy in disease states in which the endogenous levels of TNF are elevated.
  • the present invention consists in a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
  • the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
  • the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
  • the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 .' substantially prevented from binding to naturally occurring biologically active ligands.
  • sequence regions are topographically represented in Figure 26.
  • the present invention consists in a method of treating viral infection in a mammal comprising administering to the animal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to residues 1 to 18 of human TNF.
  • the present invention consists in a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153.
  • the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26.
  • the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
  • the ligand is monoclonal antibody designated MAb 32.
  • MAb 32 A sample of the hybridoma producing MAb 32 was deposited with the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom on 3 August 1989 and was accorded accession number 89080302.
  • the method of treatment includes the co-administration of another anti-viral agent, such as, IL-2, AZT or acyclovir.
  • another anti-viral agent such as, IL-2, AZT or acyclovir.
  • the present inventors have also found that there is a synergistic effect in treatment of viral infection between gamma interferon and TNF to which the ligand of the present is bound. This synergistic effect can be obtained either by the administration of the anti-TNF ligand alone by making use of endogenous TNF and endogenous interferon. As would be clear to a person skilled in the art this same effect may be obtained in the following ways:- 1. Administration of anti-TNF ligand bound to TNF (making use of endogenous interferon);
  • the method of treatment includes the co-administration of gamma interferon with the anti-TNF ligand either alone or in combination with TNF.
  • the present invention consists in a composition for use in treating viral infection the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
  • the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
  • the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
  • the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands.
  • sequence regions are topographically represented in Figure 26.
  • the present invention consists in a composition for use in treating viral infection in a mammal the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to residues 1 - 18 of human TNF.
  • the present invention consists in a composition for use in treating viral infection in a mammal comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153.
  • the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26.
  • the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301) .
  • the ligand is monoclonal antibody designated MAb 32.
  • the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
  • the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
  • the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
  • the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands.
  • sequence regions are topographically represented in Figure 26.
  • the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the anti-TNF ligand being characterised in that it binds to residues 1 - 18 of human TNF.
  • the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153.
  • the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26.
  • the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
  • the ligand is monoclonal antibody designated MAb 32.
  • the ligand is selected from the group consisting of antibodies, F(ab) fragments, restructured antibodies (CDR grafted humanised antibodies), single domain antibodies (dABs), single chain antibodies, anti-idiotypic antibodies, serum binding proteins, receptors and natural inhibitors.
  • the ligand may also be a protein or peptide which has been synthesised and which is analogous to one of the foregoing fragments. However, it is presently preferred that the ligand is a monoclonal or polyclonal antibody or F(ab) fragment thereof.
  • TNF TNF Regression
  • Endothelial Procoagulant "Induction of Tumour Fibrin Deposition”, “Cytotoxicity” and “Receptor Binding” are to be determined by the methods described below.
  • single domain antibodies as used herein is used to denote those antibody fragments such as described in Ward et al (Nature, Vol. 341, 1989, 544 - 546) as suggested by these authors.
  • Fig. 1 shows the results of a titration assay with MAb 32 against TNF
  • Fig. 2 shows the effect of anti-TNF monoclonal antibodies 1 and 32 on TNF cytotoxicity in WEHI-164 cells;
  • Fig. 3 shows the effect of anti-TNF MAbs on induction of endothelial cell procoagulant activity by TNF;
  • Fig. 4 is a schematic representation of epitopes on TNF;
  • Fig. 5 shows binding of radio labelled TNF to receptors on bovine aortic endothelial cells
  • Fig. 6 shows receptor binding studies of TNF complexed with MAb 32 (— — ) , control antibody (. and MAb 47 ( ⁇ fl
  • Fig. 7 shows receptor binding studies of TNF co plexed with MAb 32 (- _. ), control antibody (— Eh- ) and MAb 47 (— ⁇ —) on melanoma cell line IGR3;
  • Fig. 8 shows receptor binding studies of TNF complexed with MAb 32 (— —), control antibody (— Q—) and MAb 47 (— ⁇ —) on bladder carcinoma cell line 5637;
  • Fig. 9 shows receptor binding studies of TNF complexed with MAb 32 ( - ) , control antibody (—Q—) and MAb 47 (— f —) on breast carcinoma cell line MCF7;
  • Fig. 10 shows receptor binding studies of TNF complexed with MAb 32 (— — ⁇ ) .
  • Fig. 11 shows the effect on TNF-mediated tumour regression in vivo by MAb 32 ( gg ) control MAb ( ⁇ g ) and MAb 47 (*);
  • Fig. 12 shows the effect on TNF-mediated tumour regression in vivo by control MAb, MAb 32 and univalent FAb' fragments of MAb 32;
  • Fig. 13 shows the effect on TNF induced tumour regression by control MAb ( flf ) , MAb 32 ( Qg . ) and peptide 301 antiserum ( £y ) ;
  • Fig. 14 shows MAb 32 reactivity with overlapping peptides of 10 AA length
  • Fig. 15 shows a schematic three dimensional representation of the TNF molecule
  • Fig. 16 shows topographically the region of residues 1 - 26, 117 - 128 and 141 - 153;
  • Fig. 17 shows the virus levels in ovaries following treatment with TNF alone and with 200 ⁇ l TN -MAb 32; O 2 ⁇ g TNF; f 2 / jg TNF + Ab; ⁇ 4 ⁇ g TNF; ⁇ 4 ⁇ g TNF + Ab;
  • Fig. 18 shows the virus levels in lungs following administration of TNF alone and 200 ⁇ l TNF-MAb 32; D 2 ⁇ g TNF; M 2 ⁇ g TNF + Ab; ⁇ 4 ⁇ g TNF; 4 ⁇ g TNF + Ab; Fig. 19 shows virus levels in spleens following administration of TNF alone and 200 ⁇ l TNF-MAb 32; O 2 ⁇ g TNF; _f 2 ⁇ g TNF + Ab; 2 4 ⁇ g TNF; gg 4 ⁇ g TNF + Ab; Fig.
  • Fig. 21 shows in vitro anti-HSV-1 induction in L929 cells treated with TNF and MAb 32;
  • Fig. 22 shows HSV-1 titration in the ovaries of mice treated twenty-four hours before infection with various TNF concentrations with or without Ab301; CD TNF alone; ⁇ 2 TNF plus 1/50 Ab 301; and
  • Fig. 23 shows the binding of I-TNF to L929 cells either alone or in the presence of MAb 32 or Ab 301;
  • TNF alone O TNF plus MAb 32; — ⁇ — TNF plus AB 301. Animals and Tumour Cell Lines
  • mice were immunised with 10 ug human recombinant TNF intra-peritoneally in Freund's complete adjuvant. One month later 10 ug TNF in Freund's incomplete adjuvant was administered. Six weeks later and four days prior to fusion selected mice were boosted with 10 ug TNF in PBS. Spleen cells from immune mice were fused with the myeloma Sp2/0 according to the procedure of Rathjen and Underwood (1986, Mol. Immunol. 2 441). Cell lines found to secrete anti-TNF antibodies by radioimmunoassay were subcloned by limiting dilution on a feeder layer of mouse peritoneal irtacrophages. Antibody subclasses were determined by ELISA (Misotest, Commonwealth Serum Laboratories). Radioimmunoassay
  • TNF was iodinated using lac operoxidase according to standard procedures. Culture supernatants from hybridomas (50 ul) were incubated with 1251 TNF (20,000 cpm in 50 ul) overnight at 4°C before the addition of 100 ul Sac-Cel (donkey anti-mouse/rat immunoglobulins coated cellulose, Wellcome Diagnostics) and incubated for a further 20 minutes at room temperature (20 C) . Following this incubation 1 ml of PBS was added and the tubes centrifuged at 2,500 rpm for 5 minutes. The supernatant was decanted and the pellet counted for bound radioactivity. Antibody-Antibody Competition Assays
  • the comparative specificites of the monoclonal antibodies were determined in competition assays using either immobilized antigen (LACT) or antibody (PACT) (Aston and Ivanyi, 1985, Pharmac. Therapeut. 22, 403) PACT
  • tumour regression activity was assessed in three tumour models: the subcutaneous tumours WEHI-164 and Meth A sarcoma and the ascitic Meth A tumour.
  • Subcutaneous tumours were induced by the injection of approximately 5 x
  • tumours between 10 - 15 mm approximately 14 days later.
  • Mice were injected intra-peritoneally with human recombinant TNF (10 micrograms) plus monoclonal antibody (200 microlitres ascites globulin) for four consecutive days.
  • Control groups received injections of PBS alone or TNF plus monoclonal antibody against bovine growth hormone.
  • tumour size was measured with calipers in the case of solid tumours or tumour-bearing animals weighed in the case of ascites mice. These measurements were taken daily throughout the course of the experiment. Radio-Receptor Assays
  • WEHI-164 cells grown to confluency were scrape harvested and washed once with 1% BSA in Hank's balanced salt solution (HBSS, Gibco) .
  • 100 ul of unlabelled TNF (1-10,000 ng/tube) or monoclonal antibody (10 fold dilutions commencing 1 in 10 to 1 in 100,000 of ascitic globulin) was added to 50ul 1251 TNF (50,000 cpm).
  • WEHI cells were then added (200 microlitres containing 2 x 10 cells) . This mixture was incubated in a shaking water bath at 37°C for 3 hours. At the completion of this incubation 1 ml of HBSS was added and the cells spun at 16,000 rpm for 30 seconds. The supernatant was discarded and bound 1251 TNF in the cell pellet counted. All dilutions were prepared in HBSS containing 1% BSA. Procoagulant Induction by TNF on Endothelial Cells
  • Bovine aortic endothelial cells (passage 10) were grown in RPMI-1640 containing 10% foetal calf serum (FCS), penicillin, streptomycin, and 2-mercaptoethanol at 37°C in 5% C0 2 .
  • FCS foetal calf serum
  • penicillin penicillin
  • streptomycin 2-mercaptoethanol
  • the cells were trypsinised and plated into 24-well Costar trays according to the protocol of Bevilacqua et al. f 1986 (PNAS £1, 4533).
  • TNF (0-500 units/culture) and monoclonal antibody (1 in 250 dilution of ascitic globulin) was added after washing of the confluent cell monolayer with HBSS. After 4 hours the cells were scrape harvested, frozen and sonicated.
  • Total cellular procoagulant activity was determined by the recalcification time of normal donor platelet-poor plasma performed at 37 C, 100 microlitres of citrated platelet-poor plasma was added to 100 ul of cell lysate and 100 ul of calcium chloride (30mM) and the time taken for clot formation recorded.
  • tumour cell culture supernatant was added to endothelial cells treated with TNF and/or monoclonal antibody (final concentration of 1 in 2) .
  • MAbs 1, 47 and 54 which have been shown in competition binding studies to share an epitope on TNF, can be seen to have highly desirable characteristics in treatment of toxic shock and other conditions of bacterial, viral and parasitic infection where TNF levels are high requiring complete neutralisation of TNF.
  • Other monoclonal antibodies such as MAb 32 are more appropriate as agents for coadministration with TNF during cancer therapy since they do not inhibit tumour regression but do inhibit activation of coagulation. This form of therapy is particularly indicated in conjunction with cytotoxic drugs used in cancer therapy which may potentiate activation of coagulation by TNF (e.g.
  • MAb 32 (Fig. 1) is an IgG2b,K antibody with an
  • MAb 32 does not inhibit TNF cytotoxicity in vitro as determined in the WEHI-164 assay.
  • Monoclonal antibody 32 variably enhances TNF-induced tumour regression activity against WEHI-164 fibrosarcoma tumours implanted subcutaneously into BALB/c mice at a TNF dose of lOug/day (see Fig. 11). This feature is not common to all monoclonal antibodies directed against TNF but resides within the binding site specificity of MAb 32 (Fig. 4) which may allow greater receptor mediated uptake of TNF into tumour cells (see Table 3).
  • Enhancement of TNF activity by MAb 32 at lower doses of TNF is such that at least tenfold less TNF is required to achieve the same degree of tumour regression (see Fig. 11.
  • the results for day 1, 2.5ug and lug TNF and day 2, 5ug, 2.5ug and lug are statistically significant in a t-test at p ( .01 level. This level of enhancement also increases the survival rate of recipients since the lower dose of TNF used is not toxic.
  • Fig. 12 shows that univalent Fab fragments of MAb 32 also cause enhancement of TNF-induced tumour regression in the same manner as whole MAb 32 (see below).
  • MAb 32 inhibits the expression of clotting factors on endothelial cells normally induced by incubation of the cultured cells with TNF (see Fig. 3). This response may be mediated by a previously unidentified TNF receptor which is distinct to the receptor found on other cells.
  • MAb 32 enhances the in vivo activation of coagulation within the tumour bed as shown by the incorporation of radiolabelled fibrinogen. This may be due to activation of monocytes/macrophage procoagulant and may provide further insight into the mechanism of TNF-induced tumour regression.
  • the BAE cells were incubated for one hour in the presence of either cold TNF (0 to lOOng) or MAb (ascites globulins diluted 1/100 to 1/100,000) and iodinated TNF (50,000 cpm) . At the end of this time the medium was withdrawn and the cells washed before being lysed with IM sodium hydroxide. The cell lysate was then counted for bound radioactive TNF. Specific binding of labelled TNF to the cells was then determined.
  • results obtained in the clotting assay using BAE cells cultured in the presence of TNF and anti-TNF MAb correlate with the results obtained in the BAE radioreceptor assay i.e. MAbs which inhibit the induction of clotting factors on the surface of endothelial cells (as shown by the increase in clotting time compared to TNF alone) also inhibit the binding of TNF to its receptor. This is exemplified by MAbs 32 and 47.
  • MAb 32 which does not inhibit TNF binding to WEHI-164 cells, does inhibit binding of TNF to endothelial cells. This result provides support for the hypothesis that distinct functional sites exist on the TNF molecule and that these sites interact with distinct receptor subpopulations on different cell types. Thus ligands which bind to defined regions of TNF are able to modify the biological effects of TNF by limiting its binding to particular receptor subtypes. As shown in Figure 5 MAb 47 is a particularly potent inhibitor of TNF interaction with endothelial cells, the percentage specific binding at a dilution of 1/100 to 1/10,000 being effectively zero. RECEPTOR BINDING STUDIES OF HUMAN TNF COMPLEXED WITH MAB 32 ON HUMAN CARCINOMA CELL LINES IN VITRO
  • MAb 32 has been shown to enhance the anti-tumour activity of human TNF.
  • the mechanisms behind the enhancement may include restriction of TNF binding to particular (tumour) receptor subtypes but not others (endothelial) with subsequent decrease in TNF toxicity to non-tumour cells. This mechanism does not require enhanced uptake of TNF by tumour cells in in vitro assays.
  • MAb 32 also potentiates the binding of human TNF directly to TNF receptors on certain human carcinoma cell lines.
  • the following human carcinoma cell lines have been assayed for enhanced receptor-mediated uptake of TNF in the presence of MAb 32: BIO, CaCo, HT 29, SKC01 (all colon carcinomas), 5637 (Bladder carcinoma), MM418E (melanoma), IGR3 (melanoma), MCF 7 (breast carcinoma).
  • the cells were propogated in either RPMI-1640 (MM418E) DMEM (CaCo and IGR 3) or Iscoves modified DMEM (BIO, HT 29, SK01, S637, MCF 7) supplemented with 10% foetal calf serum, penecillin/streptomycin and L-glutamine.
  • Receptor assays were performed as previously described for endothelial cells except that the incubation time with iodinated TNF was extended to 3 hours for all but the BIO cells for which the radiolabel was incubated for 1 hour.
  • MAb32 did not affect TNF-receptor interaction in any of the other cell lines as shown by B 10 (Fig. 10)
  • MAb 47 which has been shown to inhibit TNF binding to WEHI-164 cells and endothelial cells, and which also inhibits TNF-mediated tumour regression was found to markedly inhibit TNF binding to all the cell lines tested (Figs. 6-10).
  • Receptor binding analyses have indicated a second mechanism whereby MAb 32 may potentiate the anti-tumour activity of TNF.
  • This second pathway for enhancement of TNF results from increased uptake of TNF by tumour all receptors in the presence of MAb 32.
  • MAB 32 OR UNIVALENT FAB' FRAGMENTS OF MAB 32
  • Fig. 12 The results using the univalent FAb' fragments of MAb 32 are shown in Fig. 12. Tumour size was determined daily during the course of the experiment. The results show the mean +/- SD% change in tumour area at the completion of treatment (day 2). Differences between the control, TNF and MAb 32-TNF treated groups are statistically significant in a T-test at the p ( .01 level.
  • Fig. 13 shows the percent change in tumour area in tumour-bearing mice treated for three days with TNF plus control MAb (antibody against bovine growth hormone), TNF plus MAb 32 or TNF plus antiserum (globulin fraction) against peptide 301.
  • control group is significantly different from both of the test groups (MAb 32, antiserum 301) while the MAb 32 and peptide antiserum 301 groups are not significantly different from each other, (control vs MAb 32, p ( .002; control vs antipeptide 301, p ( .025).
  • antisera raised using a peptide which comprises part of the MAb 32 specificity also causes TNF enhancement of tumour regression.
  • the peptides were tested for reactivity with the MAbs by ELISA.
  • MAbs which had TNF reactivity absorbed from them by prior incubation with whole TNF were also tested for reactivity with the peptides and acted as a negative control.
  • Longer peptides of TNF were synthesized as described below. These peptides were used to raise antisera in sheep using the following protocol.
  • Peptide 304 H-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-
  • Peptide 309 H-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-
  • PepSyn KA is a polydimethylacrylamide gel on Kieselguhr support with 4-hydroxymethylphenoxy- acetic acid as the functionalised linker (Atherton et al., 1975, J.Am.Chem. Soc. JLZ, 6584-6585).
  • the carboxy terminal amino acid was attached to the solid support by a DCC/DMAP-mediated symmetrical-anhydride esterification.
  • Peptide 301, 302, 305 are cleaved form the resin with 95% TFA and 5% thioanisole (1.5 h) and purified on reverse phase C4 column, (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A) .
  • Peptide 303, 304 are cleaved from the resin with 95% TFA and 5% phenol (5-6 h) and purified on reverse phase C4 column. (Buffers as above).
  • Peptide 306, 308 are cleaved from the resin with 95% TFA and 5% water (1.5 h) and purified on reverse phase C4 column. (Buffers as above) .
  • Peptide 309 Peptide was cleaved from the resin with 95% TFA and 5% thioanisole and purified on reverse phase C4 column. (Buffers as above) .
  • Peptide 307 Peptide was cleaved from the resin with a mixture of 93% TFA, 3.1% Anisole, 2.97% Ethylmethylsulfide and 0.95% Ethanedithiol (3 h) and purified on reverse phase C4 column. (Buffers as above) .
  • Typical results of MAb ELISA using the 7 and 10 mers are shown in Fig. 21. Together with the results of PACT assays using the sheep anti-peptide sera (shown in Table 6) the following regions of TNF contain the binding sites of the anti-TNF MAbs.
  • MAbs in group I MAbs 1, 21, 47, 54, 37, 32 and 25
  • MAbs in group II of the schematic diagram MAbs 11, 12, 53 and 42
  • MAbs which inhibit the induction of endothelial cell procoagulant activity (MAbs 1, 32, 42, 47, 54 and 53) all bind in the region of residues 108-128 which again contains a loop structure in the 3-D model and may indicate that this region interacts with TNF receptors which are found on endothelial cells but not tumour cells.
  • MAb 32 which potentiates the in vivo tumour regression and anti-viral activity of TNF is the only antibody which binds all the loop regions associated with residues 1-26, 117-128, and 141-153 and hence binding of these regions is crucial for enhanced TNF bioactivity with concommittant reduction of toxicity for normal cells.
  • MAb 1, 47 and 54 have the same effect on the bioactivity of TNF.
  • a ligand which binds to TNF predominately in the regions of residues 22-40 and 69-97 will have the same effect on bioactivity of TNF as MAb 12.
  • a ligand which binds to TNF predominately in the regions of residues 1-30, 117-128, and 141-153 would be expected to have the same effect on the bioactivity of TNF as MAb 32 and a ligand which binds to TNF predominately in the regions of residues 22-40, 49-97, 110-127 and 136-153 would be expected to have the same effect on the bioactivity of TNF as MAb 42.
  • a ligand which binds to TNF predominately in the regions of residues 22-31 and 146-157 would be expected to have the same effect on the bioactivity of TNF as MAb 37 and a ligand which binds to TNF predominately in the regions of residues 22-40, 69-97, 105-128 and 135-155 would be e: ectt i to have the same effect on the bioactivity of TNF as MAb 53.
  • the bioactivity of TNF can be altered by the binding of a ligand to the TNF, and that the effect on the bioactivity is a function of the specificity of the ligand.
  • the binding of MAb 32 to TNF in the regions of residues 1-26, 117-128 and 141-153 results in the induction of endothelial procoagulant activity of the TNF and binding of TNF to receptors on endothelial cells being inhibited; the induction of tumour fibrin deposition and tumour regression activities of the TNF being enhanced; the cytotoxicity being unaffected and the tumour receptor binding activities of the TNF being unaffected or enhanced.
  • this effect on the bioactivity of the TNF may be due to the prevention of the binding of the epitope of the TNF recognised by MAb 32 to naturally occurring biologically active ligands. Accordingly, it is believed that a similar effect to that produced by MAb 32 could also be produced by a ligand which binds to a region of TNF in a manner such that the epitope recognised by MAb 32 is prevented from binding to naturally occurring biologically active ligands. This prevention of binding may be due to steric hindrance or other mechanisms.
  • TNF has been shown to exert an anti-viral effect both in vitro (Mestan et al Nature 323, 816-819, 1986; Wong and Goeddel Nature 323, 819-822, 1986) and in vivo (Doherty et al J.Immunol. 142, 376-380, 1989).
  • the present inventors investigated the effect of TNF-MAb 32 complexes on the anti-viral effect of TNF in vaccinia infected mice.
  • mice Twenty-four hours prior to infection of CBA-H mice with vaccinia (10 PFU W-HA-TK, Ramshaw et al Nature 329, 44-46, 1987) the mice were treated with either TNF alone (recombinant human TNF) or TNF and MAb 32 (200 ul ascites globulin) which had been mixed twenty minutes prior to inoculation.
  • Virus titres in ovaries, lung and spleen samples which had been homogenised and treated with trypsin (1 mg/ml) were determined four days later using the 143B indicator cell line.
  • Mice which were treated with TNF-MAb 32 showed reduced virus levels in ovaries (Fig. 17), lungs (Fig. 35), and spleen (Fig. 19) compared to mice treated with TNF alone.
  • mice were treated 24 hours before infection with 107 pfu Herpes Simplex Virus 1 (HSV-1) (ip) with the relevant TNF +/- Ab 301 administrations.
  • HSV-1 Herpes Simplex Virus 1
  • the antibody was diluted 1/50 before mixing with 6.0 micrograms of TNF, and the mixture then left for an hour at room temperature. From this stock TNF+Ab 301 the various concentration sof TNF in complex with the Ab was removed and diluted in PBS (i.e., 0.5-2.0 micrograms).
  • PBS i.e., 0.5-2.0 micrograms
  • the mice were left for three days post infection after which the animals were sacrificed and the ovaries aseptically removed. These organs were then homogenised in PBS and 100 ⁇ l treated with 0.1% trypsin for 30 minutes. Trypsinisation was stopped by the addition of FCS.
  • L929 cells were seeded at a concentration of 500 000 cells/well (Linbro 24 well plate) in the presence of TNF alone (10-400 ng) or in complex with Mab 32 (complex procedure described above) . These cultures were then left for 24 hours after which they were infected with HSV-1 (0.1-2.0 MOI). 100 ⁇ l of virus-containing PBS was left to absorb on the cells for 1 hour at 370C, then the excess virus was removed and the cells overlaid with F15 +5% FCS. These cultures were then left for 48 hours. After this period the cells were frozen and thawed x2 and the supernatents serially diluted (trypsin treatment not necessary) and absorbed and grown on Vero cells as described above. The results are shown in Fig. 21.
  • the administration of TNF in combination with the anti-TNF ligand MAb 32 results in a decrease in the number of virus particles recoverable from the infected animal.
  • the enhanced anti-viral effect provided by the administration of TNF in combination with an anti-TNF ligand is a result of the ligand increasing the amount of TNF available by either preventing the binding of the TNF to endothelial receptors or by directly increasing the binding of TNF to receptors on virus infected cells (Fig. 23).
  • the method of the present invention would be particularly applicable to the treatment of viral infections which are not confined to the infection of endothelial surfaces.
  • the method of the present invention is applicable in the treatment of infection with the following viruses, hepatitis, AIDS, herpes, viral meningitis, green monkey virus and vaccinia.

Abstract

Procédé de traitement d'une infection virale chez un mammifère. Le procédé consiste à administrer au mammifère un ligand anti-facteur alpha de nécrose tumorale (TNF), le ligand étant seul ou associé au TNF. Le ligand anti-TNF est caractérisé en ce que, lorsqu'il se lie au TNF, l'activité biologique de celui-ci se modifie. On a également prévu une composition destinée au traitement des infections virales chez un mammifère.A method of treating a viral infection in a mammal. The method involves administering to the mammal an anti-tumor necrosis factor (TNF) alpha ligand, the ligand being alone or associated with TNF. The anti-TNF ligand is characterized in that, when it binds to TNF, its biological activity changes. A composition is also provided for the treatment of viral infections in a mammal.

Description

METHOD OF TREATING VIRAL INFECTION Field of the Invention
The present invention relates to a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with tumour necrosis factor alpha (TNF) . The ligand is characterised in that the binding of the ligand to the TNF is such that the biological activity of TNF is modified. The invention also relates to a composition for treating viral infection.
Background of the Invention
Tumor necrosis factor alpha (TNF) is a product of activated macrophages first observed in the serum of experimental animals presensitized with Bacillus Calmette-Guerin or Corynebacterium parvum and challenged with endotoxin (LPS) . Following the systematic administration of TNF hae orrhagic necrosis was observed in some transplantable tumours of mice while in vitro TNF caused cytolytic or cytostatic effects on tumour cell lines. In addition to its host-protective effect, TNF has been implicated as the causative agent of pathological changes in septicemia, cachexia and cerebral malaria. Passive immunization of mice with a polyclonal rabbit serum against TNF has been shown to protect mice against the lethal effects of LPS endotoxin, the initiating agent of toxic shock, when administered prior to infection.
The gene encoding TNF has been cloned allowing the usefulness of this monokine as a potential cancer therapy agent to be assessed. While TNF infusion into cancer patients in stage 1 clinical trials has resulted in tumour regression, side-effects such as thro bocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension have also been reported. These quite significant side-effects associated with the clinical use of TNF are predictable in view of the many known effects of TNF, some of which are listed in Table 1. TABLE 1 BIOLOGICAL ACTIVITIES OF TNF -ANTI-TϋMOUR -ANTI-VIRAL -ANTI-PARASITE
FUNCTION cytotoxic action on tumour cells pyrogenie activity angiogenic activity inhibition of lipoprotein lipase activation of neutrophils osteoclast activation induction of endothelial, monocyte and tumour cell procoagulant activity induction of surface antigens on endothelial cells induction of IL-6 induction of c-myc and c-fos induction of EGF receptor induction of IL-1 induction of TNF synthesis induction of GM-CSF synthesis increased prostaglandin and collagenase synthesis induction of acute phase protein C3
Of particular importance is the activation of coagulation which occurs as a consequence of TNF activation of endothelium and also peripheral blood monocytes. Disseminated intravascular coagulation is associated with toxic shock and many cancers including gastro-intestinal cancer, cancer of the pancreas, prostate, lung, breast and ovary, melanoma, acute leukaemia, myeloma, myeloproliferative syndrome and myeloblastic leukaemia. Clearly modifications of TNF activity such that tumour regression activity remains intact but other undesirable effects such as activation of c agulation are removed or masked would lead to a more advantageous cancer therapy. Complete abrogation of TNF activity is sought for successful treatment of toxic shock. Segregation of hormonal activity through the use of site-specific antibodies (both polycloηal and monoclonal) can result in enhanced hormonal activity (Aston et al, 1989, Mol. Immunol. .26., 435). To date few attempts have been made to assign antigenicity or function to particular regions of the TNF molecule for which the three-dimensional structure is now known. Assignment of function to such regions would permit the development of MAbs and other ligands of therapeutic use. Polyclonal antibodies to amino acids 1 to 15 have been reported to block Hela R19 cell receptor binding by TNF (Socher et al, 1987, PNAS 84f 8829) whilst monoclonal antibodies recognising undefined conformational epitopes on TNF have been shown to inhibit TNF cytotoxicity in vitro (Bringman and Aggarwal, 1987, Hybridoma 6., 489). However, the effects of these antibodies on other TNF activities is unknown. Description of the Present Invention
The present inventors have produced panels of monoclonal antibodies active against human TNF and have characterised them with respect to their effects on the anti-tumour effect of TNF (both in vitro and in vivo) , TNF receptor binding, activation of coagulation (both in vitro and in vivo) and defined their topographic specificities. This approach has led the inventors to show that different topographic regions of TNF alpha are associated with different activities. This work is described in detail in a co-pending patent application filed under the Patent Cooperation Treaty in the Australian Receiving Office on 7 August 1990 and disclosure of this application is incorporated herein by reference. The present inventors have made the surprising finding that the administration of TNF in combination with a specific anti-TNF ligand provides an effective anti-viral treatment. In addition, it is believed that the administration of the specific anti-TNF ligand alone will provide an effective anti-viral therapy as the ligand will bind to endogenous TNF, thereby providing the same effect as if the anti-TNF ligand was administered in combination with TNF. This administration of the anti-TNF ligand alone may be the preferred method of therapy in disease states in which the endogenous levels of TNF are elevated. Summary of the Invention
Accordingly, in a first aspect the present invention consists in a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced. In a preferred embodiment of the present invention the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
In a further preferred embodiment of the present invention the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
In yet a further preferred embodiment of the present invention the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 .' substantially prevented from binding to naturally occurring biologically active ligands. Such sequence regions are topographically represented in Figure 26.
In a second aspect the present invention consists in a method of treating viral infection in a mammal comprising administering to the animal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to residues 1 to 18 of human TNF.
In a third aspect the present invention consists in a method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153. In a preferred embodiment of this aspect of the present invention the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26. In a preferred embodiment of the present invention the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
In a preferred embodiment of the present invention ' the ligand is monoclonal antibody designated MAb 32. A sample of the hybridoma producing MAb 32 was deposited with the European Collection of Animal Cell Cultures (ECACC), Vaccine Research and Production Laboratory, Public Health Laboratory Service, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom on 3 August 1989 and was accorded accession number 89080302.
In a preferred embodiment of the present invention the method of treatment includes the co-administration of another anti-viral agent, such as, IL-2, AZT or acyclovir. The present inventors have also found that there is a synergistic effect in treatment of viral infection between gamma interferon and TNF to which the ligand of the present is bound. This synergistic effect can be obtained either by the administration of the anti-TNF ligand alone by making use of endogenous TNF and endogenous interferon. As would be clear to a person skilled in the art this same effect may be obtained in the following ways:- 1. Administration of anti-TNF ligand bound to TNF (making use of endogenous interferon);
2. Anti-TNF ligand together with gamma interferon (making use of endogenous TNF); and
3. Administration of anti-TNF ligand bound to TNF together with gamma interferon.
Accordingly, in a preferred embodiment of the present invention the method of treatment includes the co-administration of gamma interferon with the anti-TNF ligand either alone or in combination with TNF. In a fourth aspect the present invention consists in a composition for use in treating viral infection the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
In a preferred embodiment of the present invention the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
In a further preferred embodiment of the present invention the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
In yet a further preferred embodiment of the present invention the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands. Such sequence regions are topographically represented in Figure 26. In a fifth aspect the present invention consists in a composition for use in treating viral infection in a mammal the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to residues 1 - 18 of human TNF.
In a sixth aspect the present invention consists in a composition for use in treating viral infection in a mammal comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153.
In a preferred embodiment of this aspect of the present invention the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26.
In a preferred embodiment of the present invention the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301) .
In a preferred embodiment of the present invention the ligand is monoclonal antibody designated MAb 32.
In a seventh aspect the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
In a preferred embodiment of the present invention the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced. In a further preferred embodiment of the present invention the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands. In yet a further preferred embodiment of the present invention the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands. Such sequence regions are topographically represented in Figure 26.
In an eighth aspect the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the anti-TNF ligand being characterised in that it binds to residues 1 - 18 of human TNF. In a ninth aspect the present invention consists in the use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153.
In a preferred embodiment of this aspect of the present invention the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153. Such sequence regions are topographically represented in Figure 26.
In a preferred embodiment of the present invention the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
In a preferred embodiment of the present invention the ligand is monoclonal antibody designated MAb 32. In a preferred embodiment of all aspects of the present invention the ligand is selected from the group consisting of antibodies, F(ab) fragments, restructured antibodies (CDR grafted humanised antibodies), single domain antibodies (dABs), single chain antibodies, anti-idiotypic antibodies, serum binding proteins, receptors and natural inhibitors. The ligand may also be a protein or peptide which has been synthesised and which is analogous to one of the foregoing fragments. However, it is presently preferred that the ligand is a monoclonal or polyclonal antibody or F(ab) fragment thereof.
The biological activities of TNF referred to herein by the terms "Tumour Regression", "Induction of
Endothelial Procoagulant", "Induction of Tumour Fibrin Deposition", "Cytotoxicity" and "Receptor Binding" are to be determined by the methods described below.
The term "single domain antibodies" as used herein is used to denote those antibody fragments such as described in Ward et al (Nature, Vol. 341, 1989, 544 - 546) as suggested by these authors.
Further information regarding anti-idiotypic antibodies can be found in Gaulton, G.N. and Greane, M.I. 1986. Idiotypic mimicry of biological receptors, Ann. Rev. Immunol. 4., 253-280; Sege, K and Peterson, P. ., 1978. Use of anti-idiotypic antibodies as cell surface receptor probes. Proc. Natl. Acad. Sci. U.S.A. 75f 2443-2447). In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following example and accompanying figures in which:-
Fig. 1 shows the results of a titration assay with MAb 32 against TNF;
Fig. 2 shows the effect of anti-TNF monoclonal antibodies 1 and 32 on TNF cytotoxicity in WEHI-164 cells; Fig. 3 shows the effect of anti-TNF MAbs on induction of endothelial cell procoagulant activity by TNF; Fig. 4 is a schematic representation of epitopes on TNF;
Fig. 5 shows binding of radio labelled TNF to receptors on bovine aortic endothelial cells;
Fig. 6 shows receptor binding studies of TNF complexed with MAb 32 (— — ) , control antibody (. and MAb 47 (~fl| ) on melanoma cell line MM418E;
Fig. 7 shows receptor binding studies of TNF co plexed with MAb 32 (- _. ), control antibody (— Eh- ) and MAb 47 (—■—) on melanoma cell line IGR3; Fig. 8 shows receptor binding studies of TNF complexed with MAb 32 (— —), control antibody (— Q—) and MAb 47 (—■—) on bladder carcinoma cell line 5637;
Fig. 9 shows receptor binding studies of TNF complexed with MAb 32 ( - ) , control antibody (—Q—) and MAb 47 (— f —) on breast carcinoma cell line MCF7; Fig. 10 shows receptor binding studies of TNF complexed with MAb 32 (— —~) . control antibody (— Q —) and MAb 47 (—β-—) on colon carcinoma cell line BIO; Fig. 11 shows the effect on TNF-mediated tumour regression in vivo by MAb 32 ( gg ) control MAb ( ζ g ) and MAb 47 (*);
Fig. 12 shows the effect on TNF-mediated tumour regression in vivo by control MAb, MAb 32 and univalent FAb' fragments of MAb 32; Fig. 13 shows the effect on TNF induced tumour regression by control MAb ( flf ) , MAb 32 ( Qg. ) and peptide 301 antiserum ( £y ) ;
Fig. 14 shows MAb 32 reactivity with overlapping peptides of 10 AA length; Fig. 15 shows a schematic three dimensional representation of the TNF molecule;
Fig. 16 shows topographically the region of residues 1 - 26, 117 - 128 and 141 - 153;
Fig. 17 shows the virus levels in ovaries following treatment with TNF alone and with 200 μl TN -MAb 32; O 2 μg TNF; f 2 /jg TNF + Ab; ^ 4 μg TNF; ^ 4 μg TNF + Ab;
Fig. 18 shows the virus levels in lungs following administration of TNF alone and 200 μl TNF-MAb 32; D 2 μg TNF; M 2 μg TNF + Ab; ^ 4 μg TNF; 4 μg TNF + Ab; Fig. 19 shows virus levels in spleens following administration of TNF alone and 200 μl TNF-MAb 32; O 2 μg TNF; _f 2 μg TNF + Ab; 2 4 μg TNF; gg 4 μg TNF + Ab; Fig. 20 shows the titration of virus from the ovaries of TNF plus 1/500 Ab 301 treated CBA/H mice four days after infection; Q PBS (N=9); 0 2μg TNF plus 1/500 AB 301 (N=7); fflj 4μg TNF plus 1/500 AB 301 (N=7); j≡[ 6μg TNF plus 1/500 AB 301 (N=4); ® 8μg TNF plus 1/500 AB 301 (N=4);
Fig. 21 shows in vitro anti-HSV-1 induction in L929 cells treated with TNF and MAb 32;
Fig. 22 shows HSV-1 titration in the ovaries of mice treated twenty-four hours before infection with various TNF concentrations with or without Ab301; CD TNF alone; β2 TNF plus 1/50 Ab 301; and
Fig. 23 shows the binding of I-TNF to L929 cells either alone or in the presence of MAb 32 or Ab 301;
TNF alone; O TNF plus MAb 32; — Φ— TNF plus AB 301. Animals and Tumour Cell Lines
In all experiments BALB/C female mice aged 10-12 weeks obtained from the CSIRO animal facility were used. Meth A solid tumour and Meth A ascites tumour cell lines were obtained from the laboratory of Dr. Lloyd J. Old (Sloan Kettering Cancer Centre) and the WEHI-164 fibrosarcoma line was obtained from Dr. Geeta Chauhdri (John Curtin School of Medical Research, Australian National University) . Fusions and Production of Hybridomas
Mice were immunised with 10 ug human recombinant TNF intra-peritoneally in Freund's complete adjuvant. One month later 10 ug TNF in Freund's incomplete adjuvant was administered. Six weeks later and four days prior to fusion selected mice were boosted with 10 ug TNF in PBS. Spleen cells from immune mice were fused with the myeloma Sp2/0 according to the procedure of Rathjen and Underwood (1986, Mol. Immunol. 2 441). Cell lines found to secrete anti-TNF antibodies by radioimmunoassay were subcloned by limiting dilution on a feeder layer of mouse peritoneal irtacrophages. Antibody subclasses were determined by ELISA (Misotest, Commonwealth Serum Laboratories). Radioimmunoassay
TNF was iodinated using lac operoxidase according to standard procedures. Culture supernatants from hybridomas (50 ul) were incubated with 1251 TNF (20,000 cpm in 50 ul) overnight at 4°C before the addition of 100 ul Sac-Cel (donkey anti-mouse/rat immunoglobulins coated cellulose, Wellcome Diagnostics) and incubated for a further 20 minutes at room temperature (20 C) . Following this incubation 1 ml of PBS was added and the tubes centrifuged at 2,500 rpm for 5 minutes. The supernatant was decanted and the pellet counted for bound radioactivity. Antibody-Antibody Competition Assays
The comparative specificites of the monoclonal antibodies were determined in competition assays using either immobilized antigen (LACT) or antibody (PACT) (Aston and Ivanyi, 1985, Pharmac. Therapeut. 22, 403) PACT
Flexible microtitre trays were coated with monoclonal antibody (sodium sulphate precipitated globulins from mouse ascites fluid, 100 micrograms per ml in sodium bicarbonate buffer, 0.05M, pH 9.6) overnight at 4°C prior to blocking non-specific binding sites with 1% bovine serum albumin in PBS (BSA/PBS). The binding of 1251 TNF to immobilised antibody was determined in the presence of varying concentrations of a second anti-TNF monoclonal antibody. Antibody and TNF were added simultaneously and incubated for 24 hours prior to washing with PBS (4 times) and counting wells for bound radioactivity. 100% binding was determined in the absence of heterologous monoclonal antibody while 100% competition was determined in the presence of excess homologous monoclonal antibody. All dilutions were prepared in BSA/PBS.
LACT
The binding of protein A purified, radiolabelled monoclonal antibodies to TNF coated microtitre wells was determined in the presence of varying concentrations of a second monoclonal antibody. Microtitre plates were coated with TNF (50 micrograms per ml) as described above. Quantities of competing antibodies (50 microlitres) were pre-incubated on plates for 4 hour at 4°C prior to addition of 1251 monoclonal antibody (30,000 cpm) for a further 24 hours. Binding of counts to wells was determined after four washes with PBS. 100% binding was determined in the absence of competing antibody while 100% competition was determined in the presence of excess unlabelled monoclonal antibody. WEHI-16 Cytotoxicity Assay
Bioassay of recombinant TNF activity was performed according to Espevik and Nissen-Meyer (1986, J. Immunol. Methods £5, 99). The effect of the monoclonal antibody on TNF activity was determined by the addition of the monoclonal antibody to cell cultures at ABT90. Tumour Regression Experiments
Modulation of TNF-induced tumour regression activity by monoclonal antibodies was assessed in three tumour models: the subcutaneous tumours WEHI-164 and Meth A sarcoma and the ascitic Meth A tumour. Subcutaneous tumours were induced by the injection of approximately 5 x
5 10 cells. This produced tumours of between 10 - 15 mm approximately 14 days later. Mice were injected intra-peritoneally with human recombinant TNF (10 micrograms) plus monoclonal antibody (200 microlitres ascites globulin) for four consecutive days. Control groups received injections of PBS alone or TNF plus monoclonal antibody against bovine growth hormone. At the commencement of each experiment tumour size was measured with calipers in the case of solid tumours or tumour-bearing animals weighed in the case of ascites mice. These measurements were taken daily throughout the course of the experiment. Radio-Receptor Assays
WEHI-164 cells grown to confluency were scrape harvested and washed once with 1% BSA in Hank's balanced salt solution (HBSS, Gibco) . 100 ul of unlabelled TNF (1-10,000 ng/tube) or monoclonal antibody (10 fold dilutions commencing 1 in 10 to 1 in 100,000 of ascitic globulin) was added to 50ul 1251 TNF (50,000 cpm). WEHI cells were then added (200 microlitres containing 2 x 10 cells) . This mixture was incubated in a shaking water bath at 37°C for 3 hours. At the completion of this incubation 1 ml of HBSS was added and the cells spun at 16,000 rpm for 30 seconds. The supernatant was discarded and bound 1251 TNF in the cell pellet counted. All dilutions were prepared in HBSS containing 1% BSA. Procoagulant Induction by TNF on Endothelial Cells
Bovine aortic endothelial cells (passage 10) were grown in RPMI-1640 containing 10% foetal calf serum (FCS), penicillin, streptomycin, and 2-mercaptoethanol at 37°C in 5% C02. For induction of procoagulant activity by TNF the cells were trypsinised and plated into 24-well Costar trays according to the protocol of Bevilacqua et al. f 1986 (PNAS £1, 4533). TNF (0-500 units/culture) and monoclonal antibody (1 in 250 dilution of ascitic globulin) was added after washing of the confluent cell monolayer with HBSS. After 4 hours the cells were scrape harvested, frozen and sonicated. Total cellular procoagulant activity was determined by the recalcification time of normal donor platelet-poor plasma performed at 37 C, 100 microlitres of citrated platelet-poor plasma was added to 100 ul of cell lysate and 100 ul of calcium chloride (30mM) and the time taken for clot formation recorded. In some experiments tumour cell culture supernatant was added to endothelial cells treated with TNF and/or monoclonal antibody (final concentration of 1 in 2) . Incorporation of 1251 Fibrinogen into Tumours of Mice
Treated with TNF and Monoclonal Antibody
In order to examine the effect of TNF and monoclonal antibodies on fibrin formation in vivof BALB/c mice were
5 injected subcutaneously with WEHI-164 cells (10 cells/animal). After 7 - 14 days, when tumours reached a size of approximately 1 cm in diameter, animals were injected intra-peritoneally with TNF (10 ug/animal) and 1251 human fibrinogen (7.5ug/animal, 122uCi/mg Amersham) either alone or in the presence of monoclonal antibody to human TNF (200ul/animal ascitic globulin). Monoclonal antibody against bovine growth hormone was used as control monoclonal antibody. Two hours after TNF infusion incorporation of 1251 fibrinogen into mouse tissue was determined by removing a piece of tissue, weighing it and counting the sample in a gamma counter.
In all 13 monoclonal antibodies reacting with human TNF were isolated. These monoclonal antibodies were designated MAb 1, MAb 11, MAb 12, MAb 20, MAb 21, MAb 25, MAb 31, MAb 32, MAb 37, MAb 42, MAb 47, MAb 53 and MAb 54. The effect of these monoclonal antibodies on the bioactivity of human TNF is set out in Table 2.
As can be seen from Table 2, whilst some monoclonal antibodies inhibit both anti-tumour activity and activation of coagulation by human TNF (MAb 1, 47 and 54) not all antibodies which inhibit the anti-tumour activity inhibit activation of coagulation either in vitro or in vivo (MAb 11, 12, 25 and 53). Indeed MAb 21 which inhibited tumour regression enhanced the activation of coagulation in vivo. TABLE 2 EFFECT OF MONOCLONAL ANTIBODIES ON TNF BIOACTIVITY
MONOCLONAL ANTIBODY
TNF BIOACTIVITY 1 11 12 20 21 25 31 32 37 42 47 53 54
Cytotoxicity _ _ _ 0 - - 0 0 0 0 - - -
Tumour
Regression _ _ _ 0 - - 0 + 0 0 - - -
Induction of Procoagulant - 0 0 - - 0 0 - o - - - - (Endothelial)
Fibrin
Deposition _ _ _ + + + + + o - - 0 -
(tumour) Receptor
Binding __ __ __ o - - 0 +/0* 0 0 - - -
(WEHI-164)
+ Enhancement 0 No effect Inhibition * Depending on MAb concentration in the case of WEHI-164 tumour cells and tumour type (see Figs. 3, 13 - 17). MAbs 1, 47 and 54, which have been shown in competition binding studies to share an epitope on TNF, can be seen to have highly desirable characteristics in treatment of toxic shock and other conditions of bacterial, viral and parasitic infection where TNF levels are high requiring complete neutralisation of TNF. Other monoclonal antibodies such as MAb 32 are more appropriate as agents for coadministration with TNF during cancer therapy since they do not inhibit tumour regression but do inhibit activation of coagulation. This form of therapy is particularly indicated in conjunction with cytotoxic drugs used in cancer therapy which may potentiate activation of coagulation by TNF (e.g. vinblastin, acyclovir, IFN alpha, IL-2, actinomycin D, AZT, radiotherapy, adriamycin, mytomycin C, cytosine arabinoside, dounorubicin, cis-platin, vincristine, 5-flurouracil, bleomycin, (Watanabe N et al 1988
Immunopharmacol. Immunotoxicol. .10.117-127) or in diseases where at certain stages TNF levels are low (e.g. AIDS) and where individuals may have AIDS associated cancer e.g. Kaposi sarcoma, non-Hodgkins lymphoma and squamous cell carcinoma.
MAb 32 (Fig. 1) is an IgG2b,K antibody with an
Q affinity for human TNF alpha of 8.77 x 10 moles/litre as determined by Scatchard analysis. This monoclonal antibody does not react with either human TNF beta (lymphotoxin) or mouse TNF alpha.
As shown in Figure 2 MAb 32 does not inhibit TNF cytotoxicity in vitro as determined in the WEHI-164 assay. Monoclonal antibody 32 variably enhances TNF-induced tumour regression activity against WEHI-164 fibrosarcoma tumours implanted subcutaneously into BALB/c mice at a TNF dose of lOug/day (see Fig. 11). This feature is not common to all monoclonal antibodies directed against TNF but resides within the binding site specificity of MAb 32 (Fig. 4) which may allow greater receptor mediated uptake of TNF into tumour cells (see Table 3).
TABLE 3
BINDING OF TNF TO RECEPTORS ON WEHI-164 CELLS IN THE PRESENCE OF MAb 32
% BINDING125 I-TNF
Enhancement of TNF activity by MAb 32 at lower doses of TNF is such that at least tenfold less TNF is required to achieve the same degree of tumour regression (see Fig. 11. The results for day 1, 2.5ug and lug TNF and day 2, 5ug, 2.5ug and lug are statistically significant in a t-test at p ( .01 level. This level of enhancement also increases the survival rate of recipients since the lower dose of TNF used is not toxic. Fig. 12 shows that univalent Fab fragments of MAb 32 also cause enhancement of TNF-induced tumour regression in the same manner as whole MAb 32 (see below).
MAb 32 inhibits the expression of clotting factors on endothelial cells normally induced by incubation of the cultured cells with TNF (see Fig. 3). This response may be mediated by a previously unidentified TNF receptor which is distinct to the receptor found on other cells.
Conversely, MAb 32 enhances the in vivo activation of coagulation within the tumour bed as shown by the incorporation of radiolabelled fibrinogen. This may be due to activation of monocytes/macrophage procoagulant and may provide further insight into the mechanism of TNF-induced tumour regression.
The results obtained with MAb 32 are shown in comparison to other anti-TNF MAbs in Table 2. The ability of MAb 32 and MAb 47 to inhibit the binding of TNF to endothelial cells was also assessed. Bovine aortic endothelial (BAE) cells (passage 11) were plated in 24-well culture dishes (Corning) which had been pre-coated with gelatin (0.2%) and grown to confluence in McCoys 5A (modified) medium supplemented with 20% foetal calf serum. For the radio-receptor assay all dilutions (of cold TNF and MAbs) were made in this medium. The BAE cells were incubated for one hour in the presence of either cold TNF (0 to lOOng) or MAb (ascites globulins diluted 1/100 to 1/100,000) and iodinated TNF (50,000 cpm) . At the end of this time the medium was withdrawn and the cells washed before being lysed with IM sodium hydroxide. The cell lysate was then counted for bound radioactive TNF. Specific binding of labelled TNF to the cells was then determined.
The results obtained in this assay with MAb 32, MAb 47 and a control MAb are set out in Figure 5.
The results obtained in the clotting assay using BAE cells cultured in the presence of TNF and anti-TNF MAb correlate with the results obtained in the BAE radioreceptor assay i.e. MAbs which inhibit the induction of clotting factors on the surface of endothelial cells (as shown by the increase in clotting time compared to TNF alone) also inhibit the binding of TNF to its receptor. This is exemplified by MAbs 32 and 47.
MAb 32, which does not inhibit TNF binding to WEHI-164 cells, does inhibit binding of TNF to endothelial cells. This result provides support for the hypothesis that distinct functional sites exist on the TNF molecule and that these sites interact with distinct receptor subpopulations on different cell types. Thus ligands which bind to defined regions of TNF are able to modify the biological effects of TNF by limiting its binding to particular receptor subtypes. As shown in Figure 5 MAb 47 is a particularly potent inhibitor of TNF interaction with endothelial cells, the percentage specific binding at a dilution of 1/100 to 1/10,000 being effectively zero. RECEPTOR BINDING STUDIES OF HUMAN TNF COMPLEXED WITH MAB 32 ON HUMAN CARCINOMA CELL LINES IN VITRO
MAb 32 has been shown to enhance the anti-tumour activity of human TNF. The mechanisms behind the enhancement may include restriction of TNF binding to particular (tumour) receptor subtypes but not others (endothelial) with subsequent decrease in TNF toxicity to non-tumour cells. This mechanism does not require enhanced uptake of TNF by tumour cells in in vitro assays. In as___ition, MAb 32 also potentiates the binding of human TNF directly to TNF receptors on certain human carcinoma cell lines. MATERIALS AND METHODS
The following human carcinoma cell lines have been assayed for enhanced receptor-mediated uptake of TNF in the presence of MAb 32: BIO, CaCo, HT 29, SKC01 (all colon carcinomas), 5637 (Bladder carcinoma), MM418E (melanoma), IGR3 (melanoma), MCF 7 (breast carcinoma). The cells were propogated in either RPMI-1640 (MM418E) DMEM (CaCo and IGR 3) or Iscoves modified DMEM (BIO, HT 29, SK01, S637, MCF 7) supplemented with 10% foetal calf serum, penecillin/streptomycin and L-glutamine. Receptor assays were performed as previously described for endothelial cells except that the incubation time with iodinated TNF was extended to 3 hours for all but the BIO cells for which the radiolabel was incubated for 1 hour. RESULTS
Enhanced TNF uptake was observed in the presence of MAb32 by the melanoma cell lines tested MM418E and IGR 3 (Figs. 13 and 14), the bladder carcinoma 5637 (Fig. 8), and the breast carcinoma MCF 7 (Fig. 9). MAb 32 did not affect TNF-receptor interaction in any of the other cell lines as shown by B 10 (Fig. 10) MAb 47, which has been shown to inhibit TNF binding to WEHI-164 cells and endothelial cells, and which also inhibits TNF-mediated tumour regression was found to markedly inhibit TNF binding to all the cell lines tested (Figs. 6-10). CONCLUSIONS
Receptor binding analyses have indicated a second mechanism whereby MAb 32 may potentiate the anti-tumour activity of TNF. This second pathway for enhancement of TNF results from increased uptake of TNF by tumour all receptors in the presence of MAb 32.
ENHANCEMENT OF TNF-MEDIATED TUMOUR REGRESSION IN VIVO BY
MAB 32 OR UNIVALENT FAB' FRAGMENTS OF MAB 32
Tumour regression studies were carried out as described above in mice carrying WEHI-164 subcutaneous tumours (N = 5 animals/group) . Tumour size was determined daily during the course of the experiment. The results obtained using MAb 32 are set out in Fig. 11 and show the mean +/- SD% change in tumour area at the completion of treatment (day 2) ( MAb 32: control MAb: *MAb 47). Differences observed between control MAb-TNF and MAb 32-TNF treated groups are statistically significant in a T-test at the p ( .01 level.
The results using the univalent FAb' fragments of MAb 32 are shown in Fig. 12. Tumour size was determined daily during the course of the experiment. The results show the mean +/- SD% change in tumour area at the completion of treatment (day 2). Differences between the control, TNF and MAb 32-TNF treated groups are statistically significant in a T-test at the p ( .01 level.
TNF INDUCED TUMOUR REGRESSION : EFFECT OF ANTI-PEPTIDE 301 SERA
Fig. 13 shows the percent change in tumour area in tumour-bearing mice treated for three days with TNF plus control MAb (antibody against bovine growth hormone), TNF plus MAb 32 or TNF plus antiserum (globulin fraction) against peptide 301. In an unpaired T-test the control group is significantly different from both of the test groups (MAb 32, antiserum 301) while the MAb 32 and peptide antiserum 301 groups are not significantly different from each other, (control vs MAb 32, p ( .002; control vs antipeptide 301, p ( .025). Thus antisera raised using a peptide which comprises part of the MAb 32 specificity, also causes TNF enhancement of tumour regression. As shown in Fig. 4 competition binding studies has s wrfn chat the thirteen monoclonal antibodies can be sub-divided into two main groups, namely MAbs 1, 21, 47, 54, 37, 32 and 25 and MAbs 11, 12, 53 and 42. Experiments were then conducted to identify the regions on human TNF recognised by these monoclonal antibodies. IDENTIFICATION OF REGIONS ON HUMAN TNF RECOGNISED BY MONOCLONAL ANTIBODIES Methods 1. Overlapping peptides of 7 and 10 amino acid residues long were synthesized on polypropylene pins according to the method of Geysen et al., 1984, PNAS 81, 3998-4002. The overlap was of 6 and 9 residues respectively and collectively the peptides covered the entire TNF amino acid sequence. The peptides were tested for reactivity with the MAbs by ELISA. MAbs which had TNF reactivity absorbed from them by prior incubation with whole TNF were also tested for reactivity with the peptides and acted as a negative control. 2. Longer peptides of TNF were synthesized as described below. These peptides were used to raise antisera in sheep using the following protocol. Merino sheep were primed with TNF peptide conjugated to ovalbumin and emulsified in Freunds Complete adjuvant and boosted at 4 weekly intervals with peptide-ovalbumin and sera assayed for the presence of anti-TNF antibody by radioimmunoassay. Of the peptides shown only peptides 275, 301, 305, 306 and 307 elicited sera reacting with whole TNF. The positive sera were then used in competitive binding assays (PACT assays) with the MAbs. The following peptides were synthesised and are described using the conventional three letter code for each amino acid with the TNF sequence region indicated in brackets. Peptide 275
H-Ala-Lys-Pro-Trp-Tyr-Glu-Pro-Ile-Tyr-Leu-OH (111-120)
Peptide 301
H-Val-Arg-Ser-Ser-Ser-Arg-Thr-Pro-Ser-Asp-Lys-Pro-Val-Ala- His-Val-Val-Ala-OH (1-18)
Peptide 302
H-Leu-Arg-Asp-Asn-Gln-Leu-Val-Val-Pro-Ser-Glu-Gly-Leu-Tyr-
Leu-Ile-OH (43-58)
Peptide 304 H-Leu-Phe-Lys-Gly-Gln-Gly-Cys-Pro-Ser-Thr-His-Val-Leu-Leu-
Thr-His-Thr-Ile-Ser-Arg-Ile-OH (63-83)
Peptide 305
H-Leu-Ser-Ala-Glu-Ile-Asn-Arg-Pro-Asp-Tyr-Leu-Asp-Phe-Ala-
Glu-Ser-Gly-Gln-Val-OH (132-150) Peptide 306
H-Val-Ala-His-Val-Val-Ala-Asn-Pro-Gln-Ala-Glu-Gly-Gln-Leu-
OH (13-26)
Peptide 307
H-Ala-Glu-Gly-Gln-Leu-Gln-Trp-Leu-Asn-Arg-Arg-Ala-Asn-Ala- Leu-Leu-Ala-Asn-Gly-OH (22-40)
Peptide 308
H-Gly-Leu-Tyr-Leu-Ile-Tyr-Ser-Gln-Val-Leu-Phe-Lys-Gly-Gln-
Gly-OH (54-68)
Peptide 309 H-His-Val-Leu-Leu-Thr-His-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-
Thr-Gln-Thr-Lys-Val-Asn-Leu-Leu-COOH (73-94)
Peptide 323
H-Thr-Ile-Ser-Arg-Ile-Ala-Val-Ser-Thr-Gln-Thr-OH (79-89) These peptides were synthesised using the following general protocol.
All peptide were synthesised using the Fmoc-polyamide method of solid phase peptide synthesis (Atherton et al,
1978, J.Chem.Soc.Chem.Commun. , 13, 537-539). The solid resin used was PepSyn KA which is a polydimethylacrylamide gel on Kieselguhr support with 4-hydroxymethylphenoxy- acetic acid as the functionalised linker (Atherton et al., 1975, J.Am.Chem. Soc. JLZ, 6584-6585).
The carboxy terminal amino acid was attached to the solid support by a DCC/DMAP-mediated symmetrical-anhydride esterification.
All Fmoc-groups were removed by piperidine/DMF wash and peptide bonds were formed either via pen afluorophenyl active esters or directly by BOP/NMM/HOBt (Castro's reagent) (Fournier et al, 1989, Int.J.Peptide Protein Res., 21, 133-139) except for certain amino acids as specified in Table 4.
Side chain protection chosen for the amino acids was removed concomittantly during cleavage with the exception of Acm on cysteine which was left on after synthesis.
TABLE 4
Peptide 301, 302, 305 are cleaved form the resin with 95% TFA and 5% thioanisole (1.5 h) and purified on reverse phase C4 column, (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A) .
Peptide 303, 304 are cleaved from the resin with 95% TFA and 5% phenol (5-6 h) and purified on reverse phase C4 column. (Buffers as above).
Peptide 306, 308 are cleaved from the resin with 95% TFA and 5% water (1.5 h) and purified on reverse phase C4 column. (Buffers as above) .
Peptide 309 Peptide was cleaved from the resin with 95% TFA and 5% thioanisole and purified on reverse phase C4 column. (Buffers as above) .
Peptide 307 Peptide was cleaved from the resin with a mixture of 93% TFA, 3.1% Anisole, 2.97% Ethylmethylsulfide and 0.95% Ethanedithiol (3 h) and purified on reverse phase C4 column. (Buffers as above) . RESULTS
Typical results of MAb ELISA using the 7 and 10 mers are shown in Fig. 21. Together with the results of PACT assays using the sheep anti-peptide sera (shown in Table 6) the following regions of TNF contain the binding sites of the anti-TNF MAbs.
MAb 1 residues 1-18, 58-65, 115-125, 138-149 MAb 11 residues 49-98 MAb 12 residues 22-40, 70-87 MAb 21 residues 1-18, 76-90 MAb 25 residues 12-22, 36-45, 96-105, 132-157 MAb 32 residues 1-26, 117-128, 141-153 MAb 37 residues 22-31, 146-157 MAb 42 residues 22-40, 49-96, 110-127, 136-153 MAb 47 residues 1-18, 108-128 MAb 53 residues 22-40, 69-97, 105-128, 135-155 MAb 54 residues 56-79, 110-127, 136-155
TABLE 5
COMPETITIVE BINDING OF TNF BY ANTI-TNF MONOCLONES IN THE PRESENCE OF ANTI PEPTIDE SERA
MAB/PEPTIDE SERA 275 301 305 306 307
I - +
II - +/- -
12 + - - ++
21 - ++ 25 - + - - -
32 - _-+++ + +
37 - + +/- - +
47 - +
53 - + - - + 54 - +
42 - + + - +
Note 1: - indicates no competition, + indicates slight competition at high concentration of anti-peptide antisera (1/50), +_-_-_- indicates strong competition by anti-peptide sera equal to that of the homologous MAb. Note 2: Only peptides which elicited sera recognising whole TNF were used in this assay. Antibody 301
Sheep were immunised with Peptide 301 conjugated with ovalbumin in Freunds adjuvant. Antiserum reactive with Peptide 301 was obtained from immunised sheep. CONCLUSIONS
Mapping of the regions recognised by each of the MAbs has indicated that MAbs in group I (MAbs 1, 21, 47, 54, 37, 32 and 25) as shown on the schematic diagram bind TNF in the region of residues 1-18 with the exception of MAbs 37 and 54, while MAbs in group II of the schematic diagram (MAbs 11, 12, 53 and 42) bind TNF in the region of residues 70-96 which encompasses a so-called pallendromic loop on the TNF 3-D structure. MAbs which inhibit the induction of endothelial cell procoagulant activity (MAbs 1, 32, 42, 47, 54 and 53) all bind in the region of residues 108-128 which again contains a loop structure in the 3-D model and may indicate that this region interacts with TNF receptors which are found on endothelial cells but not tumour cells. MAb 32 which potentiates the in vivo tumour regression and anti-viral activity of TNF is the only antibody which binds all the loop regions associated with residues 1-26, 117-128, and 141-153 and hence binding of these regions is crucial for enhanced TNF bioactivity with concommittant reduction of toxicity for normal cells. As is apparent from Table 2 MAb 1, 47 and 54 have the same effect on the bioactivity of TNF. From the results presented above it is noted that these three monoclonals bind to similar regions of the TNF molecule. Accordingly, it is believed that a ligand which binds to TNF in at least two regions selected from the group consisting predominately of the region of residues 1-20, the region of residues 56-77, the region of residues 108-128 and the region of residues 138-149 will effect the bioactivity of TNF in a manner similar to that of MAbs 1, 47 and 54. Similarly, it is believed that a ligand which binds to TNF predominately in the regions of residues 1-20 and 76-90 will have the same effect on the bioactivity of TNF as MAb 21. A ligand which binds to TNF predominately in the regions of residues 22-40 and 69-97 will have the same effect on bioactivity of TNF as MAb 12. A ligand which binds to TNF predominately in the regions of residues 1-30, 117-128, and 141-153 would be expected to have the same effect on the bioactivity of TNF as MAb 32 and a ligand which binds to TNF predominately in the regions of residues 22-40, 49-97, 110-127 and 136-153 would be expected to have the same effect on the bioactivity of TNF as MAb 42. A ligand which binds to TNF predominately in the regions of residues 22-31 and 146-157 would be expected to have the same effect on the bioactivity of TNF as MAb 37 and a ligand which binds to TNF predominately in the regions of residues 22-40, 69-97, 105-128 and 135-155 would be e: ectt i to have the same effect on the bioactivity of TNF as MAb 53.
The present inventors have quite clearly shown that the bioactivity of TNF can be altered by the binding of a ligand to the TNF, and that the effect on the bioactivity is a function of the specificity of the ligand. For example, the binding of MAb 32 to TNF in the regions of residues 1-26, 117-128 and 141-153 results in the induction of endothelial procoagulant activity of the TNF and binding of TNF to receptors on endothelial cells being inhibited; the induction of tumour fibrin deposition and tumour regression activities of the TNF being enhanced; the cytotoxicity being unaffected and the tumour receptor binding activities of the TNF being unaffected or enhanced. It is believed that this effect on the bioactivity of the TNF may be due to the prevention of the binding of the epitope of the TNF recognised by MAb 32 to naturally occurring biologically active ligands. Accordingly, it is believed that a similar effect to that produced by MAb 32 could also be produced by a ligand which binds to a region of TNF in a manner such that the epitope recognised by MAb 32 is prevented from binding to naturally occurring biologically active ligands. This prevention of binding may be due to steric hindrance or other mechanisms.
Accordingly, it is intended that the prevention of the binding of epitopes recognised by MAb 32 and Ab 301 described herein to naturally occurring biologically active ligands is within the scope of the present invention.
Effect of Anti-TNF MAb 32 on Anti-Viral Activity of Human TNF Alpha
TNF has been shown to exert an anti-viral effect both in vitro (Mestan et al Nature 323, 816-819, 1986; Wong and Goeddel Nature 323, 819-822, 1986) and in vivo (Doherty et al J.Immunol. 142, 376-380, 1989). The present inventors investigated the effect of TNF-MAb 32 complexes on the anti-viral effect of TNF in vaccinia infected mice.
Twenty-four hours prior to infection of CBA-H mice with vaccinia (10 PFU W-HA-TK, Ramshaw et al Nature 329, 44-46, 1987) the mice were treated with either TNF alone (recombinant human TNF) or TNF and MAb 32 (200 ul ascites globulin) which had been mixed twenty minutes prior to inoculation. Virus titres in ovaries, lung and spleen samples which had been homogenised and treated with trypsin (1 mg/ml) were determined four days later using the 143B indicator cell line. Mice which were treated with TNF-MAb 32 showed reduced virus levels in ovaries (Fig. 17), lungs (Fig. 35), and spleen (Fig. 19) compared to mice treated with TNF alone.
Similar results were obtained with mice treated with TNF and antibody 301 (Fig. 20). HSV-1 In Vivo Protocol
CBA/H mice were treated 24 hours before infection with 107 pfu Herpes Simplex Virus 1 (HSV-1) (ip) with the relevant TNF +/- Ab 301 administrations. The antibody was diluted 1/50 before mixing with 6.0 micrograms of TNF, and the mixture then left for an hour at room temperature. From this stock TNF+Ab 301 the various concentration sof TNF in complex with the Ab was removed and diluted in PBS (i.e., 0.5-2.0 micrograms). The mice were left for three days post infection after which the animals were sacrificed and the ovaries aseptically removed. These organs were then homogenised in PBS and 100 μl treated with 0.1% trypsin for 30 minutes. Trypsinisation was stopped by the addition of FCS. This sample was then serially diluted by a factor of 1/10. These dilutions were then absorbed onto Vero cells for 1 hour at 370C, overlaid with F15 + 5% FCS and left for 2 days at 370C. The plaques were then counted and the virus concentration calculated. The results are shown in Fig. 22. HSV-1 In Vitro Protocol
L929 cells were seeded at a concentration of 500 000 cells/well (Linbro 24 well plate) in the presence of TNF alone (10-400 ng) or in complex with Mab 32 (complex procedure described above) . These cultures were then left for 24 hours after which they were infected with HSV-1 (0.1-2.0 MOI). 100 μl of virus-containing PBS was left to absorb on the cells for 1 hour at 370C, then the excess virus was removed and the cells overlaid with F15 +5% FCS. These cultures were then left for 48 hours. After this period the cells were frozen and thawed x2 and the supernatents serially diluted (trypsin treatment not necessary) and absorbed and grown on Vero cells as described above. The results are shown in Fig. 21.
125 The Binding of I-TNF to L929 Cells Either Alone or in the Presence of Mab 32 or Ab 301
To different dilutions of Mab 32 or Ab 301 in RPMI plus 10% FCS iodinated TNF (50 000 cpm/50 μl) was added followed by 2.0 x 10 L929 cells, again in RPMI. These treatments were then incubated for 3 hours after which the cells were washed once and the pellets counted. All treatments were done in triplicate. The results are shown in Fig. 23.
Effect of Co-Administration of Gamma Interferon on Antiviral Activity of Anti-TNF Ligand and TNF Antibody 301 complexed to TNF was produced by diluting antibody 301 1:50 prior to mixing with 6 μg TNF. The TNF plus antibody was administered (i.p.) alone as well as 10 U/mouse murine gamma interferon alone. A TNF with Ab 301 plus mIFN-y treatment was also administered. The mlFN-γ was not mixed with the TNF: Ab 301 complex until after the 1 hour room temperature incubation of TNF with the antibody. 24 hours later the g mice were injected with 10 pfu vaccinia intravenously (i.v.). The results of this experiment are shown in Table 6.
TABLE 6
As can be seen from the results set out in Table 6 there is a marked synergistic anti-viral effect between the mIFN-y and the MAb 301: TNF complex.
As is readily apparent from the results shown in Figs. 17, 18 and 19 the administration of TNF in combination with the anti-TNF ligand MAb 32 results in a decrease in the number of virus particles recoverable from the infected animal. Without wishing to be bound by scientific theory, it is believed that the enhanced anti-viral effect provided by the administration of TNF in combination with an anti-TNF ligand is a result of the ligand increasing the amount of TNF available by either preventing the binding of the TNF to endothelial receptors or by directly increasing the binding of TNF to receptors on virus infected cells (Fig. 23). Accordingly, it is believed that the method of the present invention would be particularly applicable to the treatment of viral infections which are not confined to the infection of endothelial surfaces. In particular, it is believed that the method of the present invention is applicable in the treatment of infection with the following viruses, hepatitis, AIDS, herpes, viral meningitis, green monkey virus and vaccinia.
It will be recognised by persons skilled in the art that numerous variations and modifications may be made to the invention as described above without departing from the spirit or scope of the invention as broadly described.

Claims

CLAIMS : -
1. A method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
2. A method as claimed in claim 1 in which the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
3. A method as claimed in claims 1 or 2 in which the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
4. A method as claimed in claims 1 or 2 in which the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117 - 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands.
5. A method of treating viral infection in a mammal comprising administering to the animal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to residues 1 to 18 of human TNF.
6. A method of treating viral infection in a mammal comprising administering to the mammal an anti-TNF ligand either alone or in combination with TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153.
7. A method as claimed in claim 6 in which the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153.
8. A method as claimed in claims 3 or 5 in which the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
9. A method as claimed in any one of claims 1-7 in which the ligand is monoclonal antibody designated MAb 32.
10. A method as claimed in any one of claims 1-9 in which the method of treatment includes the co-administration of another anti-viral agent, such as, IL-2, AZT or acyclovir.
11. A method as claimed in any one of claims 1-10 in which the method of treatment includes the co-administration of gamma interferon with the anti-TNF ligand either alone or in combination with TNF.
12. A composition for use in treating viral infection the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
13. A composition as claimed in claim 12 in which the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
14. A composition as claimed in claims 12 or 13 in which the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
15. A composition as claimed in claims 12 or 13 in which the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117
- 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands.
16. A composition for use in treating viral infection in a mammal the composition comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to residues 1
- 18 of human TNF.
17. A composition for use in treating viral infection in a mammal comprising gamma interferon and an anti-TNF ligand either alone or bound to TNF, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141-153.
18. A composition as claimed in claims 15 or 17 in which the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153.
19. A composition as claimed in claims 14 or 16 in which the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
20. A method as claimed in any one of claims 11-19 in which the ligand is monoclonal antibody designated MAb 32.
21. The use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that when it binds to TNF the induction of endothelial procoagulant activity of the TNF is inhibited and the anti-viral activity of the TNF is unaffected or enhanced.
22. The use as claimed in claim 21 in which the ligand is further characterised in that when it binds to TNF the binding of TNF to receptors on endothelial cells is inhibited; the induction of tumour fibrin deposition and tumour regression activities for the TNF are enhanced; the cytotoxicity is unaffected and the tumour receptor binding activities of the TNF are unaffected or enhanced.
23. The use as claimed in claims 21 or 22 in which the ligand is characterised in that the epitope of the TNF defined by the topographic region of residues 1 to 18 is substantially prevented from binding to naturally occurring biologically active ligands.
24. The use as claimed in claims 21 or 22 in which the ligand binds to TNF such that the epitope of the TNF defined by the topographic regions of residues 1 - 30, 117
- 128 and 141 - 153 and more preferably in the topographic regions of residues 1 - 26, 117 - 128 and 141-153 is substantially prevented from binding to naturally occurring biologically active ligands.
25. The use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the anti-TNF ligand being characterised in that it binds to residues 1
- 18 of human TNF.
26. The use of an anti-TNF ligand either alone or in combination with TNF in the production of a medicament for the treatment of viral infection in a mammal, the ligand being characterised in that it binds to human TNF in the topographic regions of residues 1 - 30, 117 - 128 and 141
- 153.
27. The use as claimed in claim 26 in which the ligand binds to human TNF in the topographic regions of residues 1 - 26, 117 - 128 and 141-153.
28. The use as claimed in claim 25 in which the ligand is an antibody raised against a peptide having an amino acid sequence substantially corresponding to amino acids 1 to 18 of human TNF (Peptide 301).
29. The use as claimed in an one of claims 21-28 in which the ligand is monoclonal antibody designated MAb 32.
30. A method as claimed in any one of claims 1-9 in which the ligand is selected from the group consisting of antibodies, F(ab) fragments, restructured antibodies (CDR grafted humanised antibodies), single domain antibodies (dABs), single chain antibodies, anti-idiotypic antibodies, serum binding proteins, receptors and natural inhibitors.
31. A method as claimed in claim 30 in which the ligand is a monoclonal or polyclonal antibody or F(ab) fragment thereof.
EP91915297A 1990-08-27 1991-08-27 Method of treating viral infection Ceased EP0608212A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
AU1976/90 1990-08-27
AUPK197690 1990-08-27
PCT/AU1991/000400 WO1992003145A1 (en) 1990-08-27 1991-08-27 Method of treating viral infection

Publications (2)

Publication Number Publication Date
EP0608212A4 EP0608212A4 (en) 1993-10-20
EP0608212A1 true EP0608212A1 (en) 1994-08-03

Family

ID=3774922

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91915297A Ceased EP0608212A1 (en) 1990-08-27 1991-08-27 Method of treating viral infection

Country Status (4)

Country Link
EP (1) EP0608212A1 (en)
JP (1) JPH06500323A (en)
CA (1) CA2090401A1 (en)
WO (1) WO1992003145A1 (en)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5587457A (en) * 1990-03-12 1996-12-24 Peptide Technology Limited Neutrophil stimulating peptides
US6375928B1 (en) * 1990-03-12 2002-04-23 Peptech Limited Neutrophil stimulating peptides
IT1254315B (en) * 1992-03-27 1995-09-14 Mini Ricerca Scient Tecnolog ANTI-IDIOTYPIC MONOCLONAL ANTIBODIES DIRECT AGAINST ANTI-TNF ANTIBODIES.
ATE172880T1 (en) * 1992-08-28 1998-11-15 Bayer Ag USE OF MONOCLONAL ANTI-TNF ANTIBODIES FOR THE TREATMENT OF BACTERIAL MENINGITIS
US6090382A (en) 1996-02-09 2000-07-18 Basf Aktiengesellschaft Human antibodies that bind human TNFα
CA2817619A1 (en) 2001-06-08 2002-12-08 Abbott Laboratories (Bermuda) Ltd. Methods of administering anti-tnf.alpha. antibodies
US20030206898A1 (en) 2002-04-26 2003-11-06 Steven Fischkoff Use of anti-TNFalpha antibodies and another drug
US20040033228A1 (en) 2002-08-16 2004-02-19 Hans-Juergen Krause Formulation of human antibodies for treating TNF-alpha associated disorders
MY150740A (en) 2002-10-24 2014-02-28 Abbvie Biotechnology Ltd Low dose methods for treating disorders in which tnf? activity is detrimental
EP1578799B8 (en) 2002-12-02 2011-03-23 Amgen Fremont Inc. Antibodies directed to tumor necrosis factor and uses thereof
AU2009333883B2 (en) 2008-12-29 2015-05-21 Trevena, Inc. Beta-arrestin effectors and compositions and methods of use thereof
CA2862933A1 (en) 2012-01-31 2013-08-08 Trevena, Inc. .beta.-arrestin effectors and compositions and methods of use thereof
CN106170293A (en) 2014-02-07 2016-11-30 特维娜有限公司 The crystal form of β suppression albumen effector and amorphous form
JP2017518290A (en) 2014-05-19 2017-07-06 トレベナ・インコーポレイテッドTrevena, Inc. Synthesis of β-arrestin effector

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03503840A (en) * 1988-01-15 1991-08-29 セントコアー,インコーポレーテッド Heterologous linked antibodies and their therapeutic uses
AU4182289A (en) * 1988-08-19 1990-03-23 Celltech Limited Pharmaceutical products for anti-neoplastic therapy
GB8905400D0 (en) * 1989-03-09 1989-04-19 Jonker Margreet Medicaments
GB8921123D0 (en) * 1989-09-19 1989-11-08 Millar Ann B Treatment of ards

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9203145A1 *

Also Published As

Publication number Publication date
JPH06500323A (en) 1994-01-13
CA2090401A1 (en) 1992-02-28
WO1992003145A1 (en) 1992-03-05
EP0608212A4 (en) 1993-10-20

Similar Documents

Publication Publication Date Title
AU640400B2 (en) Tumour necrosis factor binding ligands
US6593458B1 (en) Tumor necrosis factor peptide binding antibodies
US6451983B2 (en) Tumor necrosis factor antibodies
US7544782B2 (en) Tumour necrosis factor binding ligands
EP0608212A1 (en) Method of treating viral infection
AU654501B2 (en) Method of treating viral infection

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19930325

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): CH DE DK FR GB IT LI SE

17Q First examination report despatched

Effective date: 19960111

GRAG Despatch of communication of intention to grant

Free format text: ORIGINAL CODE: EPIDOS AGRA

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 19970726