WO2002034780A2 - Procedes et moyens de deoligomerisation de molecules oligomeres - Google Patents

Procedes et moyens de deoligomerisation de molecules oligomeres Download PDF

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WO2002034780A2
WO2002034780A2 PCT/EP2001/012422 EP0112422W WO0234780A2 WO 2002034780 A2 WO2002034780 A2 WO 2002034780A2 EP 0112422 W EP0112422 W EP 0112422W WO 0234780 A2 WO0234780 A2 WO 0234780A2
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subunit
protein complex
hetero
tnf
oligomeric
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PCT/EP2001/012422
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WO2002034780A3 (fr
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Paul Ameloot
Peter Brouckaert
Walter Fiers
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Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
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    • 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]
    • 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

Definitions

  • the invention relates to a method to form artificial, hetero-oligomeric complexes of proteins that do normally occur in homo-oligomeric complexes.
  • Said hetero-oligomeric complexes do have a biological activity that is similar or identical to the activity of the homo-oligomeric complexes.
  • the current invention also relates to a method to use said hetero-oligomeric complexes to screen for compounds that can stabilize said hetero-oligomeric complexes, or to screen for compounds that dissociate said hetero-oligomeric complexes into monomers, which are not biologically active, or show a biological activity, which is clearly distinct from that of the hetero-oligomeric complexes, or to screen for compounds that inhibit the oligomerisation or reoligomerisation of monomers in oligomeric complexes.
  • the invention relates to pharmaceutical compositions comprising said compounds.
  • cytokine mediated response is an essential part of the normal response to trauma and infection
  • excessive production of pro-inflammatory cytokines or production of cytokines in a wrong biological context is associated with a wide range of diseases, such as sepsis, rheumatoid arthritis, inflammatory bowel disease, cancer, response to malaria and AIDS.
  • PIGF Placenta Growth Factor
  • VEGF Vascular Endothelial Growth Factor
  • Tumour Necrosis Factor TNF
  • TNF Tumour Necrosis Factor
  • the latter family comprises 19 known members (LTA, TNF, LTB, PGL.YRP, TNFSF4, TNFSF5, TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11 , TNFSF12, TNFSF13, TNFSF13B, TNFSF14, TNFSF15, TNFSF18 (all names according to the Human Genome nomenclature database) and TNFSF20 (Tribouley et al., 1999).
  • LTA LTA
  • TNF transmembrane proteins
  • the extracellular domain has ⁇ jelly roll structure (Banner et ai, 1993) and is important in ligand trimerisation. Intrinsic to this trimerisation is the formation of a receptor-binding site at the junction between neighbouring subunits, creating a multivalent ligand.
  • ligands of the TNF superfamily are only biologically active as homotrimeric or natural heterotrimeric protein complexes and not as monomeric protein.
  • TNF TNF-binding protein
  • TNF-R1 p55 TNF-R; CD120a
  • TNF-R2 p75 TNF-R; CD120b
  • TNF homotrimeric protein complex can bind three TNF-receptor molecules. Binding of the ligand to the receptors induces a clustering of the intracellular domains, initiating the intracellular signalling cascades. As the formation of a homotrimeric protein complex is essential for the receptor binding, dissociation of the homotrimeric complex into monomeric TNF impairs its biological activity.
  • TNF TNF-binding protein
  • reagents such as Triton X-100, DMSO or guanidinium chloride promote the dissociation of TNF.
  • This dissociation can be reversed by enhancing the TNF concentration or removing the chemical agent (Hlodan and Pain, 1995; Corti et ai., 1992; Hlodan and Pain, 1995).
  • An excess of soluble TNF- receptors, which only bind to trimeric TNF, on the other hand can stabilise the biological activity of subnanomolar amounts of TNF during incubation, presumably by diminishing the dissociation of TNF (Aderka et ai, 1992).
  • lymphokines and cytokines in general and TNF in particular are involved in the pathogenesis of several inflammatory diseases, they are obvious and attractive therapeutical targets.
  • TNF inhibition by administration of anti-TNF monoclonal antibodies or soluble TNF receptors (or TNF receptor fusion proteins) is effective in the treatment of rheumatoid arthritis and inflammatory bowel disease (Camussi and Lupia, 1998; Sandborn and Hanauer, 1999).
  • Isolation of alternative compounds that can either inactivate or stabilise growth factors, lymphokines and cytokines, and particularly TNF is therefore an important aim in pharmaceutical research.
  • the activity of growth factors, lymphokines and cytokines is often measured in biological assays.
  • these assays are not suitable to screen for inhibitors, because it is unclear at which level the inhibitor is operating. Indeed, if in such an assay inhibition is detected, it can be due to an inactivation of the ligand, as well as to an inactivation of the receptor or of one of the compounds of the signalling pathway. Compounds that act on the latter levels may cause unwanted side effects.
  • the present invention describes a method to screen for compounds that can stabilise or destabilise a homo-oligomeric protein complex, by the use of a hetero-oligomeric protein complex, comprising one or more modifications of the subunit of said homo- oligomeric protein complex, whereby said hetero-oligomeric protein complex is preferentially retaining essentially the same biological activity as said homo-oligomeric protein complex.
  • Each modified subunit can be distinguished from the non-modified subunit and/or from those subunits carrying another modification.
  • the hetero- oligomeric complex can consist of two or more different kinds of modified subunits without any unmodified subunit, or it can consist of one or more different kinds of modified subunits, with one or more unmodified subunits.
  • hetero- oligomeric protein complex As long as the hetero- oligomeric protein complex is stable, selective capturing of either a modified or a non- modified subunit, e.g. by immunoprecipitation or binding on a solid substrate, will result in a capturing of the whole protein complex. However, when a destabilising compound is added, selective capturing will only capture the monomeric subunit to which it is directed and the remaining subunits will be released. The effect can be measured by measuring either the amount of units that are released, or the amount of subunits that are retained after a certain time period. As normally most of the oligomeric complexes do show a spontaneous destabilisation with time, not only destabilising compounds, which enhance the destabilisation, can be screened, but also compounds that do stabilise the complex and slow down the disaggregation.
  • One aspect of the invention is an artificial, biologically active hetero-oligomeric protein complex comprising one or more modifications of one subunit, possibly in combination with one or more unmodified forms of the same subunit, whereby said unmodified subunit can form a biologically active homo-oligomeric protein complex on its own, whereby the biological activity of said homo-oligomeric protein complex is essentially the same as that of said hetero-oligomeric protein complex, and whereby said unmodified subunit as well as said modifications have a clearly distinct biological activity as monomeric molecule, compared to the oligomeric complexes.
  • the monomeric molecules are biologically inactive.
  • said hetero-oligomeric and/or said homo-oligomeric protein complex is not a receptor and/or the oligomeric complex is not membrane bound. More preferably, said hetero-oligomeric and/or said homo-oligomeric protein complex is a ligand.
  • said hetero-oligomeric protein complex and said homo-oligomeric protein complex are composed of the same number of subunits. More preferably, both protein complexes are trimeric. Said modifications should be easily distinguishable from the non-modified form, or, if more than one modified form is used, from each other.
  • modifications can be 125 l iodinated forms, biotinylated forms, subunits carrying a tag such as a his-tag or subunits, comprising one or more amino acid changes, preferentially resulting in the creating or destruction of a specific epitope.
  • Homologous molecules showing at least 40% identities and/or 65% positives at protein level, as measured by BL-ASTP (Altschul et al., 1997), are still considered as modifications of the same subunit.
  • said homologous molecules show at least 60% identities, more preferably at least 75% identities, most preferably 80% identities.
  • a preferred embodiment is a hetero-oligomeric protein complex whereby said subunit is a subunit of a member of the TNF superfamily. Another preferred embodiment is a hetero-oligomeric protein complex whereby said subunit is a TNF subunit. A most preferred embodiment is a hetero-oligomeric complex whereby said unmodified subunit is a human TNF subunit, and said modification a murine TNF subunit, or said unmodified subunit is a murine TNF subunit, and said modification a human TNF subunit.
  • Another aspect of the invention is the use of a biologically active hetero-oligomeric protein complex comprising one or more modifications of one subunit, possibly in combination with one or more unmodified forms of the same subunit, whereby said unmodified subunit can form a biologically active homo-oligomeric protein complex on its own, whereby the biological activity of said homo-oligomeric protein complex is essentially the same as that of said hetero-oligomeric protein complex, and whereby said unmodified subunit as well as said modifications have a clearly distinct biological activity as monomeric molecule, compared to the oligomeric complexes, to screen compounds that stabilise or destabilise said hetero-oligomeric protein complex.
  • said hetero-oligomeric protein complex is an artificial hetero-oligomeric protein complex.
  • said hetero-oligomeric complex comprises a human TNF subunit as unmodified subunit, and a murine TNF subunit as modification, or a murine TNF subunit as unmodified subunit, and a human TNF subunit as modification.
  • Still another aspect of the invention is a method to screen compounds that stabilize or destabilize a biologically active hetero-oligomeric protein complex said protein complex comprising one or more modifications of one subunit, possibly in combination with one or more unmodified forms of the same subunit, whereby said unmodified subunit can form a biologically active homo-oligomeric protein complex on its own, whereby the biological activity of said homo-oligomeric protein complex is essentially the same as that of said hetero-oligomeric protein complex, and whereby said unmodified subunit as well as said modifications have a clearly distinct biological activity as monomeric molecule, compared to the oligomeric complexes, said method comprising a) binding one or more of the subunits in a specific way; b) contacting said hetero-oligomeric protein complex with one or more of said compounds; c) measuring the amount of one or more subunits present in monomeric and/or oligomeric form.
  • the binding can be covalently or not, and can be realized before the formation of the hetero-oligomeric complex, e.g. by binding a monomeric subunit to a solid substrate, followed by oligomerisation by contacting the immobilized subunit to free subunits, or after formation of the hetero-oligomeric complex, e.g. by specific binding of the non-modified subunit or one of the modifications by an antibody. Contacting said hetero-oligomeric protein complex with one or more of said compounds may be realized before or after said binding.
  • the stability can be measured, as non-limiting examples, by measuring the release of subunits from an immobilized hetero-oligomeric complex, as well as by measuring the amount of said hetero-oligomeric complex by precipitation of said complex, e.g. by immunoprecipitation, after a certain contact time with said stabilising or destabilising compound.
  • Quantification of monomers of oligomers can be realized, as non-limiting examples, by radioactivity or by immunological techniques.
  • said hetero-oligomeric protein complex is artificial.
  • a preferred embodiment is a method according to the invention whereby said hetero- oligomeric complex comprises a human TNF subunit as unmodified subunit, and a murine TNF subunit as modification, or a murine TNF subunit as unmodified subunit, and a human TNF subunit as modification.
  • Hetero-oligomeric protein complex as used here means a protein complex that consists of one or more identical subunits (modified or not) with at least one other subunit (modified or not), whereby latter subunit is clearly distinct from the former.
  • Artificial hetero-oligomeric complex means that the complex doesn't exist as such in nature.
  • said artificial hetero-oligomeric complex may comprise subunits coming from different sources such as different organisms, or it may comprise subunits carrying induced mutations, or it may comprise chemical modifications of the protein.
  • Comprising one or more modifications of one subunit means that the protein complex may comprise one or several modified forms of the same subunit, whereby the modification may be identical or different.
  • Subunit as used here can be either an unmodified or a modified subunit.
  • - Modification as used here may be any modification, as long as the modification allows a selective distinction of the modified form from the other forms of the subunit, modified by another modification or not modified.
  • Modification can be, as a non limiting example, radioactive labelling such as iodination, biotinylation, or the change of one or more amino acids preferentially leading to the loss or the generation of a specific epitope.
  • the replacement of a subunit by a homologous subunit of the same or another species is still considered as being a modification, as long as the homology is at least 40% identities and/or 60% positives, preferably 60% identities, more preferably
  • Biologically active hetero-oligomeric protein complex refers to the biological activity of the homo-oligomeric protein complex within the cell type and/or organism in which it is normally functional.
  • biologically active means that the trimeric complex can bind to the TNF receptors, as can be measured as described below.
  • Can form a biologically active homo-oligomeric protein complex means that the unmodified subunit, in absence of modified subunits, when brought in a suitable medium will form spontaneously a homo-oligomeric complex on its own, without interference of another protein, whereby said homo-oligomeric complex has a biological activity that is clearly distinct of that of the monomer.
  • Essentially identical as used here means that there is no essential change in function between the hetero-oligomeric protein complex and the homo-oligomeric protein complex within the cell type and/or organism in which the homo-oligomeric protein complex is tested, but does not exclude a change in specific activity
  • FIG. 1 Gel filtration experiment using TNF homo- and heterotrimers, in absence of destabilising compounds.
  • TNF deoligomerises during incubation at low concentration and monomers as well as trimers can be detected by gelfiltration (muTNF at ⁇ 400 pM ; huTNF at ⁇ 100 pM ).
  • Deoligomerisation of labeled muTNF can be prevented by addition of excess, unlabeled muTNF or huTNF.
  • FIG. 2 Effect of TNF concentration on the trapping of labeled, homotrimeric or heterotrimeric TNF in a RIA using the anti-human TNF antibody 61 E71 or the anti- murine TNF antibody 1 F3F3.
  • 125 l-human TNF was preincubated with different concentrations of unlabeled murine TNF (squares) or human TNF (circles). The mixture was applied to wells coated with 61 E71 , an anti-human TNF antibody (black symbols), or coated with 1 F3F3, an anti-murine TNF antibody (white symbols). Wells were washed and bound label was counted and is represented as percentage of label applied to well.
  • Figure 3 Binding of labeled huTNF/murine TNF heterotrimers on soluble murine TNF- receptors. 125 l-human TNF was incubated with unlabeled murine TNF (black symbols) or human TNF (white symbols) for 0 (circles) or 16 hours (triangles). The mixture was applied to wells coated with soluble murine TNF-R1 (panel A) or coated with soluble murine TNF-R2 (panel B). Bound label was measured in a gamma-counter.
  • Figure 4 Binding of labeled murine TNF-75/murine TNF heterotrimers on soluble murine TNF-receptors. 125 l-murine TNF-75 was incubated with unlabeled murine TNF for 24 hours and subsequently applied to wells coated with soluble murine TNF-R1 (white circles) or coated with soluble murine TNF-R2 (black triangles). Bound label was measured.
  • Figure 5 Screening assay with labeled human TNF/murine TNF heterotrimers in a RIA using the anti-murine TNF antibody 1 F3F3.
  • 125 l-human TNF was preincubated with unlabeled murine TNF and the mixture was applied to wells coated with the anti- murine TNF antibody 1 F3F3 to trap heterotrimeric TNF. After washing away unbound label, the wells were treated with different agents, as indicated in the legend. After 15 minutes, 4 or 18 hours incubation, the wells were washed and remaining label was counted and was represented as percentage of label bound at start of treatment.
  • Figure 6 Screening assay with labeled human TNF/murine TNF heterotrimers in a RIA using the anti-murine TNF antibody 1F3F3.
  • 125 l-human TNF was preincubated with unlabeled murine TNF and the mixture was applied to wells coated with the anti- murine TNF antibody 1 F3F3 to trap heterotrimeric TNF. After washing away unbound label, the wells were treated with different agents, as indicated in the legend. After 15 minutes, 4 or 18 hours incubation, the wells were washed and remaining label was counted and was represented as percentage of label bound at start of treatment.
  • FIG 7 Screening assay with labeled murine TNF/human TNF heterotrimers in a RIA using the anti-human TNF antibody 61 E71. 125 l-murine TNF was incubated with unlabeled human TNF and the mixture was ' applied to wells coated with antibody 61 E71. After washing away unbound label, the wells were treated with different agents for 15 minutes, 4 or 18 hours. After the treatment, wells were washed and the label present in the wells and in the wash-solutions was counted. The amount of label in the two fractions is represented as percentage of total label in both fractions.
  • Figure 8 Effect of TNF concentration on the formation and trapping of heterotrimeric TNF on streptavidin-coated wells.
  • 125 l-human TNF was preincubated with different concentrations of biotinylated human TNF and applied to wells coated with streptavidin. The wells were washed after 2 hours, and bound label was counted and is represented as percentage of label applied to well.
  • Figure 9 Screening assay using labeled human TNF/biotinylated human TNF. 125 l-human TNF was preincubated with biotinylated human TNF and the mixture was applied to streptavidin-coated wells. After washing away unbound label, the wells were treated with different agents. After 15 minutes, 4 or 18 hours incubation, the wells were washed and the remaining label was measured and was represented as percentage of label that was bound at start of treatment.
  • Figure 10 Gel filtration of labeled human TNF after pretreatment with different agents. Labeled human TNF was incubated with different agents, each at a concentration of 100 ⁇ g/ml, for 1 hour at 22°C.
  • Figure 12 Gel filtration of murine TNF. Labeled murine TNF was preincubated with different concentrations of unlabeled murine TNF (total TNF concentration is indicated in legend) as such or in the presence of methylene blue (MB). The mixture was chromatographed on a Sephacryl S100 column and the radioactivity in each fraction was measured and is here represented as percentage of label present in all fractions.
  • total TNF concentration is indicated in legend
  • MB methylene blue
  • E. coli-derived muTNF and huTNF were radiolabeled using the lodogen iodination agent according to the manufacturer's instructions (Pierce Chemicals, Rockford, IL).
  • the labeled proteins were separated from unincorporated radioactivity on a G-25 column (PD10, Pharmacia-LKB Biotechnology, Uppsala, Sweden) and had a specific radioactivity of 10-50 ⁇ Ci/ ⁇ g.
  • E. coli-derived muTNF and huTNF were biotinylated using a NHS-LC-biotinylation kit, according to the manufacturer's instructions (Pierce Chemicals, Rockford, IL).
  • the biotinylated proteins were separated from unincorporated NHS-LC-biotin on a G-25 column (PD10, Pharmacia-LKB Biotechnology, Uppsala, Sweden).
  • Radioimmunoassay (RIA) 96-well plates (Maxi Breakapart, Nunc) were incubated overnight at 4°C with monoclonal antibody (2.5 ⁇ g/ml; 100 ⁇ l/well) in PBS. After coating, wells were saturated for 3 h at 26°C with 150 ⁇ l/well of PBS containing 2% BSA. After washing with PBS, plates were incubated with samples containing radiolabeled TNF, diluted in PBS/BSA, (100 ⁇ l/well) for 90 min at 26°C. After washing with PBS, wells were separated and individually counted in a gamma counter.
  • the soluble (extracellular) fragment of murine TNF-R1 (smuTNF-R1) or smuTNF-R2 were produced with the baculovirus-expression system in Sf9 insect cells and partially purified.
  • Microtiter plates (Nunc-lmmuno Breakapart Module) were coated with smuTNF-R1 (0.66 ⁇ g/ml) or smuTNF-R2 (1 ⁇ g/ml) overnight at 4°C. Blocking was with PBS/2% BSA.
  • the microtiter plates were then incubated with 400 pM (20 ng/ml) 125 l- labeled TNF in the presence of different concentrations of unlabeled TNF. After 4 hours incubation at 26°C, the wells were washed with PBS/ 0.02% BSA and bound radioactivity was measured in a gamma-counter.
  • TNF heterotrimeric complexes are formed during coincubation of different TNFs
  • Radiolabeled human TNF (20 ng/ml) was coincubated with different concentrations of unlabeled murine TNF or human TNF at 26°C for 24 hours to form heterotrimeric as well as homotrimeric TNF. The preincubated mixture was then allowed to bind on wells of 96 well plates (Nunc-lmmuno Breakapart, Nunc), coated with monoclonal antibody against murine TNF (1 F3F3, R&D Systems) or with monoclonal antibody against human TNF (61 E71 , Dr. W. Buurman, University of Limburg, The Netherlands).
  • Radiolabeled human TNF (20 ng/ml) was coincubated with different concentrations of unlabeled murine TNF or human TNF at 26°C for 0 or 16 hours and subsequently allowed to bind on wells from a microtiter plate (Nunc-lmmuno Breakapart, Nunc), coated with soluble murine TNF-R1 or with soluble murine TNF-R2. After 4 hours incubation, the wells were washed with PBS/BSA (0.02%) and bound label was measured in a gamma-counter ( Figure 3). As expected on the base of the affinity of the receptors, labeled human TNF only binds to the soluble murine TNF-R1 (Tartaglia et al., 1991). After prior coincubation with unlabeled murine TNF, however, a significant amount of label, now present in heterotrimeric molecules, is retained on wells coated with soluble murine TNF-R2.
  • Receptor-specific muteins of human TNF have been made and described (Van Ostade et al., 1993). We have created a similar mutein of murine TNF that interacts specifically with murine TNF-R2. This mutein, murine TNF-75, was created by site- specific mutagenesis of murine TNF resulting in the replacement of amino acids Arg 32 with Tyr and Ala 145 with Arg.
  • Radiolabeled murine TNF-75 (20 ng/ml) was coincubated with different concentrations of unlabeled murine TNF at 26°C for 24 hours and subsequently allowed to bind on wells from a microtiter plate (Nunc-lmmuno Breakapart, Nunc), coated with soluble murine TNF-R1 or with soluble murine TNF-R2. After 4 hours incubation, the wells were washed with PBS/BSA (0.02%) and bound label was measured in a gamma-counter ( Figure 4).
  • Example 3 Heterotrimeric TNF molecules still interact with TNF-receptors: In vivo activity
  • Murine TNF was serially diluted in DMEM/FCS, or in DMEM/FCS containing murine TNF-75 (200 pM or 600 pM), and directly or after a preincubation of 24 hours at 37°C, 100 ⁇ l of the dilution samples was added to wells containing L929s fibrosarcoma cells (30000 cells/well). After incubation (18 hours at 37°C) in the presence of actinomycine D (1 ⁇ g/ml), surviving cells were measured by MTT staining. The biological activity of the samples is represented in U/ml (1 U/ml is the concentration of TNF sufficient to kill half of the cells) (Table 1).
  • the TNF-R2-specific murine TNF-75 is not cytotoxic to L929 cells at the concentrations used.
  • the apparent biological activity of murine TNF is strongly decreased upon preincubation in DMEM/FCS, due to dissociation into inactive monomers.
  • murine TNF-75 was present during the preincubation of murine TNF, the higher concentration of TNF monomers was promoting trimer formation and the decrease of murine TNF activity was reduced due to the formation of heterotrimeric, functional TNF. From the results, it is clear that heterotrimeric molecules containing two wild type TNF subunits are still able to bind to cell bound murine TNF-R1 and to induce the intracellular signalling cascade leading to L929s cytotoxicity.
  • Example 4 Development of a screening assay using human TNF/mouse TNF heterotrimeric protein complexes
  • Radiolabeled human TNF (20 ng/ml) was coincubated with unlabeled murine TNF (50 ng/ml) at 22°C for 16 hours to form heterotrimeric as well as homotrimeric TNF.
  • the preincubated mixture (100 ⁇ l/well) was then allowed to bind on wells of 96 well plates (Nunc-lmmuno Breakapart, Nunc), coated with monoclonal antibody against murine TNF (1F3F3, R&D Systems). After 90 minutes, the wells were washed with PBS/BSA (0.02%)). Hereby unlabeled murine TNF and labeled heterotrimeric TNF were retained.
  • Example 5 Development of a screening assay using biotinylated human TNF/iodinated human TNF heterotrimeric protein complexes
  • Biotinylated human TNF was prepared using a NHS-LC-biotinylation kit (Pierce, USA) and human TNF was labeled with 125 l using IODO-GEN iodination reagent (Pierce, Rockford, USA). Radiolabeled human TNF (100 ng/ml) was coincubated with different concentrations of biotinylated human TNF at 26°C for 16 hours to form heterotrimeric TNF, containing as well 125 l-labeled as biotinylated TNF subunits.
  • Radiolabeled human TNF 70 ng/ml was incubated with biotinylated human TNF (300 ng/ml) for 16 hours at 26°C and the mixture was allowed to bind on wells coated with streptavidin (0.25 ⁇ g/ml). After 120 minutes, the wells were washed with PBS/BSA (0.02%>), hereby retaining biotinylated homotrimeric (unlabeled) and heterotrimeric (labeled) human TNF.
  • Radiolabeled huTNF (200 ng/ml) was incubated in PBS/BSA or in PBS/BSA containing one of the above-mentioned agents at 100 ⁇ g/ml, for 1 hour at 22°C. After size exclusion chromatography on a Sephacryl S-100 column, the radioactivity present in the different fractions (400 ⁇ l) was measured using a gamma-counter. Radiolabeled murine TNF (80 ng/ml) was incubated for 16 hours with biotinylated murine TNF (250 ng/ml) and subsequently allowed to bind on wells coated with extravidin, a modified avidin reagent (Sigma nr E2511) (1 ⁇ g/ml).
  • the wells were incubated with PBS/BSA, methylene blue, or with a monoclonal antibody against murine TNF, i.e. 1 F3F3 or TN3. After incubation for 15 minutes, 4 h or 18 h, the wells were washed with PBS/BSA and the radioactivity remaining in the individual wells was counted in a Gamma 5500B counter (figure 11).
  • MuTNF was serially diluted in DMEM/FCS, with or without muTNF75 1 , and directly or after a preincubation of 24 h at 37°C, 100 ⁇ l was added to wells containing L929 cells 2 . After 18 h incubation in the presence of actinomycin D, surviving cells were measured by MTT staining. The biological activity of the samples is represented in U/ml. One U/ml is the concentration of TNF at which half of the cells are killed. Results are means (+/- SD) of three experiments.

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Abstract

L'invention concerne un procédé de fabrication de complexes hétéro-oligomères artificiels de protéines qui surviennent d'ordinaire dans des complexes homo-oligomères. Ces complexes hétéro-oligomères présentent une activité biologique semblable ou identique à celle des complexes homo-oligomères. L'invention concerne également un procédé d'utilisation desdits complexes hétéro-oligomères pour cribler des composés susceptibles de stabiliser lesdits complexes hétéro-oligomères, ou des composés qui dissocient les complexes hétéro-oligomères en monomères, qui ne sont pas actifs biologiquement, ou qui présentent une activité biologique, clairement distincte de celle des complexes hétéro-oligomères, ou des composés qui inhibent l'oligomérisation ou la réoligomérisation des monomères en complexes oligomères. L'invention traite enfin de compositions pharmaceutiques renfermant ces composés.
PCT/EP2001/012422 2000-10-26 2001-10-25 Procedes et moyens de deoligomerisation de molecules oligomeres WO2002034780A2 (fr)

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US7807784B2 (en) 2003-06-11 2010-10-05 The University Of Chicago Increased T-cell tumor infiltration by mutant LIGHT
US7811983B2 (en) 2003-06-11 2010-10-12 The University Of Chicago Increased T-cell tumor infiltration and eradication of metastases by mutant light
US8734795B2 (en) 2008-10-31 2014-05-27 Biogen Idec Ma Inc. Light targeting molecules and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040774A1 (fr) * 1995-06-07 1996-12-19 Biogen, Inc. Complexes de lymphotoxines modifiees utilises comme preparations pharmaceutiques
WO2001025277A1 (fr) * 1999-10-07 2001-04-12 Maxygen Aps Polypeptides antagonistes a chaine unique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040774A1 (fr) * 1995-06-07 1996-12-19 Biogen, Inc. Complexes de lymphotoxines modifiees utilises comme preparations pharmaceutiques
WO2001025277A1 (fr) * 1999-10-07 2001-04-12 Maxygen Aps Polypeptides antagonistes a chaine unique

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE CA [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; HOCHMAN, PAULA S. ET AL: "Proinflammatory responses are efficiently induced by homotrimeric but not heterotrimeric lymphotoxin ligands" retrieved from STN Database accession no. 125:245323 CA XP002195286 & J. INFLAMMATION (1996), VOLUME DATE 1995-1996, 46(4), 220-234 , 1996, *
J L BROWNING ET AL.: "Preparation and characterization of soluble recombinant heterotrimeric complexes of human lymphotoxin alpha and beta" JOURNAL OF BIOLOGICAL CHEMISTRY., vol. 271, no. 15, 12 April 1996 (1996-04-12), pages 8618-8626, XP002016900 AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD., US ISSN: 0021-9258 *
J L BROWNING ET AL.: "Signaling through the lymphotoxin beta receptor induces the death pf some adenocarcinoma cell lines" JOURNAL OF EXPERIMENTAL MEDICINE, vol. 183, no. 3, 1 March 1996 (1996-03-01), pages 867-878, XP000571445 TOKYO, JP ISSN: 0022-1007 *
P AMELOOT ET AL.: "Heterotrimers formed by tumor necrosis factors of different species or muteins" JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 29, 20 July 2001 (2001-07-20), pages 27098-27103, XP002195288 AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD., US *
SACCA, ROSALBA ET AL: "Differential activities of secreted lymphotoxin -.alpha.3 and membrane lymphotoxin -.alpha.1.beta.2 in lymphotoxin -induced inflammation: critical role of TNF receptor 1 signalin" J. IMMUNOL. (1998), 160(1), 485-491 , 1998, XP002195285 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7807784B2 (en) 2003-06-11 2010-10-05 The University Of Chicago Increased T-cell tumor infiltration by mutant LIGHT
US7811983B2 (en) 2003-06-11 2010-10-12 The University Of Chicago Increased T-cell tumor infiltration and eradication of metastases by mutant light
US9272025B2 (en) 2003-06-11 2016-03-01 The University Of Chicago Increased T-cell tumor infiltration and eradication of metastases by mutant light
US8734795B2 (en) 2008-10-31 2014-05-27 Biogen Idec Ma Inc. Light targeting molecules and uses thereof

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