EP0608212A1 - Procede de traitement des infections virales - Google Patents

Procede de traitement des infections virales

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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
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German (de)
English (en)
Other versions
EP0608212A4 (fr
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
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Filing date
Publication date
Application filed by Peptide Technology Ltd filed Critical Peptide Technology Ltd
Publication of EP0608212A4 publication Critical patent/EP0608212A4/fr
Publication of EP0608212A1 publication Critical patent/EP0608212A1/fr
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal 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.

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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.
EP91915297A 1990-08-27 1991-08-27 Procede de traitement des infections virales Ceased EP0608212A1 (fr)

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US6375928B1 (en) * 1990-03-12 2002-04-23 Peptech Limited Neutrophil stimulating peptides
US5587457A (en) * 1990-03-12 1996-12-24 Peptide Technology Limited Neutrophil stimulating peptides
IT1254315B (it) * 1992-03-27 1995-09-14 Mini Ricerca Scient Tecnolog Anticorpi monoclonali anti-idiotipici diretti contro anticorpi anti-tnf.
ATE172880T1 (de) * 1992-08-28 1998-11-15 Bayer Ag Verwendung von monoklonalen anti-tnf-antikörpern für die behandlung von bakteriellen meningitiden
US6090382A (en) 1996-02-09 2000-07-18 Basf Aktiengesellschaft Human antibodies that bind human TNFα
CA2868614A1 (fr) 2001-06-08 2002-12-08 Abbott Laboratories (Bermuda) Ltd. Methodes pour administrer des anticorps anti-tnf.alpha.
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
AU2003298816C1 (en) 2002-12-02 2010-12-16 Amgen Fremont, Inc. Antibodies directed to Tumor Necrosis Factor and uses thereof
HUE026131T2 (en) 2008-12-29 2016-05-30 Trevena Inc Beta-arresin effector and preparations and methods for their use
WO2013116312A1 (fr) 2012-01-31 2013-08-08 Trevena, Inc. Effecteurs de la ss-arrestine et compositions et procédés d'utilisation de ceux-ci
WO2015120316A1 (fr) 2014-02-07 2015-08-13 Trevena, Inc. Formes cristallines et amorphes d'un effecteur bêta-arrestine
CN106456698B (zh) 2014-05-19 2022-05-17 特维娜有限公司 β-抑制蛋白效应物的合成

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WO1990001950A1 (fr) * 1988-08-19 1990-03-08 Celltech Limited Produits pharmaceutiques pour therapie anti-neoplastique
GB8905400D0 (en) * 1989-03-09 1989-04-19 Jonker Margreet Medicaments
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