EP0759984A1 - Modulateur de recepteurs de la super-famille tnf/ngf et recepteurs oligomeres solubles de la super-famille tnf/ngf - Google Patents

Modulateur de recepteurs de la super-famille tnf/ngf et recepteurs oligomeres solubles de la super-famille tnf/ngf

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Publication number
EP0759984A1
EP0759984A1 EP95919787A EP95919787A EP0759984A1 EP 0759984 A1 EP0759984 A1 EP 0759984A1 EP 95919787 A EP95919787 A EP 95919787A EP 95919787 A EP95919787 A EP 95919787A EP 0759984 A1 EP0759984 A1 EP 0759984A1
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EP
European Patent Office
Prior art keywords
tnf
fas
cells
protein
binding
Prior art date
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Application number
EP95919787A
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German (de)
English (en)
Other versions
EP0759984A4 (fr
Inventor
David Wallach
Mark Boldin
Igor Mett
Eugene Dept. of Membrane Research VARFOLOMEEV
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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Priority claimed from IL109632A external-priority patent/IL109632A/en
Application filed by Yeda Research and Development Co Ltd filed Critical Yeda Research and Development Co Ltd
Publication of EP0759984A1 publication Critical patent/EP0759984A1/fr
Publication of EP0759984A4 publication Critical patent/EP0759984A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention is generally in the field of receptors belonging to the TNF NGF superfamily of receptors and the control of their biological functions
  • TNF/NGF superfamily of receptors includes receptors such as the p55 and p75 tumor necrosis factor receptors (TNF-Rs) and the FAS ligand receptor (also called FAS/APO1 or FAS-R and hereinafter will be called FAS- R.) and others.
  • TNF-Rs tumor necrosis factor receptors
  • FAS ligand receptor also called FAS/APO1 or FAS-R and hereinafter will be called FAS- R.
  • the present invention concerns novel proteins which bind to the intracellular domains (IC) of the p55 and p75 TNF-Rs and the Fas-R, (these intracellular domains designated p55IC, p75IC and Fas-IC, respectively) and which novel proteins are capable of modulating the function of the p55 and p75 TNF-Rs and the Fas-R.
  • IC intracellular domains
  • p55IC intracellular domains
  • Fas-IC Fas-R
  • One of the proteins capable of binding the p55IC of the intact p55-TNF-R is the p55IC itself in the form of a p55IC molecule or a po ⁇ ion thereof, such as for example, the so-called 'death domain' (DD) of the ⁇ 55IC.
  • DD 'death domain'
  • the present invention also concerns new TNF-associated effects that can be induced in cells in a ligand (TNF)-independent fashion by the intracellular domain of the p55 TNF-R (p55IC) or portions thereof.
  • TNF ligand
  • the present invention also concerns the preparation and uses of these novel p55 and p75 TNF-R-b ding proteins, and Fas-R binding proteins, referred to herein as p55IC-, p75IC- and Fas-IC- binding proteins.
  • the present invention also concerns new soluble oligomeric TNF-Rs.
  • oligomeric FAS-Rs and oligomeric receptors having a mixture of TNF-Rs and FAS-Rs, their uses, and methods for the production thereof.
  • TNF Tumor Necrosis Factor
  • TNF- ⁇ Lymphotoxin
  • TNF- ⁇ Tumor Necrosis Factor
  • TNF- ⁇ Lymphotoxin
  • TNF contributes to the defense of the organism against tumors and infectious agents and contributes to the recovery from injury.
  • TNF can be used as an anti-tumor agent in which application it binds to its receptors on the surface of tumor cells and thereby initiates the events leading to the death of the tumor cells.
  • TNF can also be used as an anti-infectious agent.
  • both TNF- ⁇ and TNF- ⁇ also have deleterious effects. There is evidence that over-production of TNF- ⁇ can play a major pathogenic role in several diseases.
  • TNF- ⁇ primarily on tr.e vasculature
  • TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia, and TNF- ⁇ was thus called cachetin.
  • cachetin TNF- ⁇ was thus called cachetin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler and Cerami, 1?S7) and as a major mediator of the damage observed in graft-versus-host reactions (Piquet et a ' :.. 1987).
  • TNF is known to be involved in the process of inflammation and in many other diseases.
  • TNF-Rs Two distinct, independently expressed, receptors, the p55 and p75 TNF-Rs, which bind both TNF- ⁇ and TNF- ⁇ specifically, initiate and/or mediate the above noted biological effects of TNF. These two receptors have structurally dissimilar intracellular domains suggesting that they signal differently (See Hohmann et al., 1989; Engelmann et al., 1990; Brockhaus et al , 1990; Leotscher et al., 1990. Schall et al., 1990; Nophar et al., 1990; Smith et al., 1990; and Heller et al.. 1990).
  • the cellular mechanisms for example, the various proteins and possibly other factors, which are involved in the intracellular signaling of the p55 an p75 TNF-Rs have yet to be elucidated (as set forth herein below, there is described for the first time, new proteins capable of binding to the p75IC and p55 IC). It is this intracellular signaling, which occurs usually after the binding of the ligand, i.e. TNF ( ⁇ or ⁇ ), to the receptor, that is responsible for the commencement of the cascade of reactions that ultimately result in the observed response of the cell to TNF.
  • TNF ligand
  • cytocidal effect of TNF in most cells studied so far, this effect is triggered mainly by the p55 TNF-R.
  • Antibodies against the extracellular domain (Jigand binding domain) of the p55 TNF-R can themselves trigger the cytocidal effect (see EP 412486) which co elates with the effectivity of receptor cross-linking by the antibodies, believed to be the first step in the generation of the intracellular signaling process.
  • TNF-R the biological function of the p55 TNF-R depends on the integrity of its intracellular domain, and accordingly it has been suggested that the initiation of intracellular signaling leading to the cytocidal effect of TNF occurs as a consequence of the association of two or more intracellular domains of the p55 TNF-R.
  • TNF ⁇ and ⁇
  • TNF occurs as a homotrimer and as such has been suggested to induce intracellular signaling via the p55 TNF-R by way of its ability to bind to and to cross-link the receptor molecules, i e cause receptor aggregation.
  • FAS-R FAS receptor
  • Fas antigen a cell-surface protein expressed in various tissues and sharing homology with a number of cell-surface receptors including TNF-R and NGF-R
  • the FAS-R mediates cell death in the form of apoptosis (Itoh et al., 1 91), and appears to serve as a negative selector of autoreactive T cells, i.e.
  • FAS-R mediates the apoptopic death of T cells recognizing self-antigens. It has also been found that mutations in the FAS-R gene (Ipr) cause a lymphoproliferation disorder in mice that resembles the human autoimmune disease systemic lupus erythematosus (SLE) (Watanabe-Fukunaga et al., 1992).
  • the ligand for the FAS-R appears to be a cell-surface associated molecule carried by, amongst others, killer T cells (or cytotoxic T lymphocytes - CTLs), and hence when such CTLs contact cells carrying FAS-R, they are capable of inducing apoptopic cell death of the FAS-R-carrying cells.
  • a monoclonal antibody has been prepared that is specific for FAS-R, this monoclonal antibody being capable of inducing apoptopic cell death in cells carrying FAS-R, including mouse cells transformed by cDNA encoding human FAS-R (Itoh et al., 1991).
  • EP 186833, EP 308378, EP 398327 and EP 412486 to regulate the deleterious effects of TNF by inhibiting the binding of TNF to its receptors using anti-TNF antibodies or by using soluble TNF receptors (being essentially the soluble extracellular domains of the receptors) to compete with the binding of TNF to the cell surface-bound TNF-Rs.
  • soluble TNF receptors being essentially the soluble extracellular domains of the receptors
  • EPO 568925 relates to a method of modulating signal transduction and/or cleavage in TNF-Rs whereby peptides or other molecules may interact either with the receptor itself or with effector proteins interacting with the receptor, thus modulating the normal functioning of the TNF-Rs.
  • EPO 568925 there is described the construction and characterization of various mutant p55 TNF-Rs, having mutations in the extracellular,, transmembranal, and intracellular domains of the ⁇ 55 TNF-R. In this way regions within the above domains of the p55 TNF-R were identified as being essential to the functioning of the receptor, i.e.
  • TNF ligand
  • TNF-effe ⁇ the binding of the ligand (TNF) and the subsequent signal transduction and intracellular signaling which ultimately results in the observed TNF-effe ⁇ on the cells.
  • TNF ligand
  • EPO 568925 A number of approaches for isolating and cloning the DNA 5 sequences encoding such proteins and peptides; for constructing expression vectors for the production of these proteins and peptides; and for the preparation of antibodies or fragments thereof which interact with the TNF-R or with the above proteins and peptides that bind various regions of the TNF-R, are also set forth in EPO 568925. However, no description is made in EPO 568925 of the actual proteins and peptides which bind to the intracellular domains of the TNF-Rs
  • TNF-Rs or FAS-R desirable to decrease the amount or the activity of TNF-Rs or FAS-R at the cell surface, while an increase in the amount or the activity of TNF-Rs or FAS-R would be desired when an enhanced TNF or FAS-R ligand effect is sought
  • promoters of both the p55 TNF-R and the p75 TNF-R have recently been sequenced and analyzed by the present inventors and a number of key sequence motifs have been found that are specific to various transcription regulating factors, 0 and as such the expression of these TNF-Rs can be controlled at their promoter level, i.e.
  • proteins which may regulate the effect of ligands belonging to the TNF/NGF superfamily such as the TNF or FAS-R ligand effect on cells, by mediation of the intracellular signaling process, which signaling is 0 probably governed to a large extent by the intracellular domains (ICs) of the receptors belonging to the TNF NGF superfamily of receptors, such as those of the TNF-Rs. i e the p55 and p75 TNF-R intracellular domains (p55IC and p75IC. respectively), as well as the FAS-IC.
  • Another aim of the invention is to provide antagonists (e.g. antibodies) to these intracellular domain-binding proteins (IC-binding proteins) which may be used to inhibit the signaling process, when desired, when such IC-binding proteins are positive signal effectors (i.e. induce signaling), or to enhance the signaling process, when desired, when such IC-binding proteins are negative signal effectors (i.e. inhibit signaling).
  • IC-binding proteins intracellular domain-binding proteins
  • Yet another aim of the invention is to use such IC-binding proteins to isolate and characterize additional proteins or factors, which may, for example, be involved further downstream in the signaling process, and/or to isolate and identify other receptors further upstream in the signaling process to which these IC-binding proteins bind (e.g other TNF-Rs or related receptors), and hence, in whose function the IC-binding proteins are also involved.
  • additional proteins or factors which may, for example, be involved further downstream in the signaling process, and/or to isolate and identify other receptors further upstream in the signaling process to which these IC-binding proteins bind (e.g other TNF-Rs or related receptors), and hence, in whose function the IC-binding proteins are also involved.
  • IC-binding proteins as antigens for the preparation of polyclonal and/or monoclonal antibodies thereto.
  • the antibodies may be used for the purification of the new IC-binding proteins from different sources, such as cell extracts or transformed cell lines.
  • these antibodies may be used for diagnostic purposes, e.g. for identifying disorders related to abnormal functioning of cellular effects mediated by receptors belonging to the TNF/NGF receptor superfamily.
  • a further aim of the invention is to provide pharmaceutical compositions comprising the above IC-binding proteins, and pharmaceutical compositions comprising the IC-binding protein antagonists, for the treatment or prophylaxis of TNF-induced or FAS ligand-induced conditions, for example, such compositions can be used to enhance the TNF or FAS ligand effect or to inhibit the TNF or FAS ligand effect depending on the above noted nature of the IC-binding protein or antagonist thereof contained in the composition.
  • TBP-I was shown to protect cells from TNF toxicity at concentrations of a few nanograms per ml and to interfere with the binding of both TNF- ⁇ and TNF- ⁇ to cells, when applied simultaneously with these cytokines. Further examination of the mechanism by which TBP-I functions revealed that TBP-I does not interact with the target cell, but rather blocks the function of TNF by binding TN specifically, thus competing for TNF with the TNF receptor.
  • TBP-I TBP-I
  • TBP-II TBP-II
  • Both proteins provide protection against the in vitro cytocidal effect of TNF and both bind TNF- ⁇ less effectively than TNF- ⁇
  • TBP-I and TBP-II appeared to have a very similar molecular size, they could clearly be distinguished from each other by lack of immunological cross reactivity, differing N-terminal amino acid sequences and differing amino acid composition.
  • soluble TNF binding proteins are monomeric and being capable of binding only one monomer of the TNF homotrimer, the natural ligand, which still permits TNF activity (i.e. incomplete neutralization) by virtue of the TNF still having two active monomers unbound by the TNF binding proteins.
  • soluble FAS-Rs soluble FAS-R ligand binding proteins
  • a so-called 'death domain' of the p55-IC (Tartaglia et al., 1993) has been disclosed, but did not show, in accordance with the present invention, that the p55-IC and the 'death domain' thereof self-associates, this self-association being primarily responsible for the signaling leading to induction of cell cytotoxis.
  • this publication is silent on the possibility of producing the soluble, oligomeric TNF-Rs, or the soluble, oligomeric Fas-Rs, or mixed oligomeric thereof, nor does it disclose other TNF-associated effects induced by the p55-IC or portions thereof, e.g. IL-8 gene expression induction, all of the present invention.
  • novel proteins which are capable of binding to either the intracellular domain of the p55 TNF-R (the p55IC -binding proteins), of the p75 TNF-R (the p75IC-binding proteins), and of the FAS-R (the FAS-IC-binding proteins).
  • These p55IC-, p75IC- and FAS-IC- binding proteins may act as mediators or modulators of the TNF or FAS-R ligand effect on cells by way of mediating or modulating the intracellular signaling process which usually occurs following the binding of TNF to the p55 and or p75 TNF-R, or the binding of the FAS-R ligand at the cell surface.
  • the p55IC and FAS-IC are capable of self association and that fragments of the p55IC and FAS-IC are similarly capable of binding to the p55 IC, particularly the so-called 'death domains (DD) within the ICs of these receptors, i.e. the p55DD and FAS-DD.
  • p55 IC and FAS-IC and their fragments also represent proteins capable of binding to the p55IC and FAS-IC and hence may be modulators of the TNF or FAS-R ligand effect on cells.
  • the nature of the binding of one of the novel proteins of the invention, the herein designated 55.1.1 protein, to the intracellular domain of p55-TNF-R has been more fully elucidated (see Example 1).
  • the present invention is based on the finding that the intracellular domain of the p 5 TNF receptor (p55-IC), a region contained therein, the so-called p55-IC 'death domain', the intracellular domain of the Fas APOl receptor (Fas-IC), and a region contained therein, the so-called Fas-IC 'death domain' are capable of self-association.
  • p55-IC p 5 TNF receptor
  • Fas-IC Fas APOl receptor
  • a soluble, oligomeric TNF receptor being a fusion product, containing at least two extracellular domains of a TNF receptor at its one end, and at its other end at least two of the above noted self-associating intracellular domains or portions thereof, which self-associate to provide an oligomer having at least two such fusion products linked together.
  • a soluble, oligomeric TNF-R is thus capable of binding two monomers of the naturally-occurring TNF homotrimer, and as such effectively neutralizes TNF activity.
  • TNF activity being desirable in all of the above mentioned conditions wherein TNF is overproduced endogenously or is administered exogenous. ⁇ ' in high doses resulting in undesirable side effects.
  • the effective binding of TNF by the soluble, oligomeric receptors of the invention may also serve to allow for the binding of exogenously added TNF and its subsequent desired slow-release in conditions where TNF is administered for its beneficial effects, e.g. in tumor therapy.
  • an oligomeric FAS-R being a fusion product, containing at least two extracellular domains of a FAS-R at its one end, and at its other end at least two of the above noted self-associating intracellular domains or portions thereof, which self- associate to provide an oligomer having at least two such fusion products linked together
  • Such an oligomeric FAS-R is thus capable of binding two monomers of the naturally occurring FAS-R ligand homotrimer, and as such effectively neutralizes FAS-R ligand activity The n e utralization of FAS-R ligand activity being desirable in all of the above mentioned conditions where excess amounts thereof are associated with undesirable side effects.
  • Such a mixed oligomer would be a mixture of the above noted fusion products containing at least one extracellular domain of a TNF-R and at least one extracellular domain of a FAS-R at its one end, and at its other end at least two of the above mentioned self-associating intracellular domains or portions thereof, which self-associate to provide a mixed oligomer having at least two such fusion products linked together.
  • Such a mixed oligomer is thus capable of binding at least one monomer of TNF and one monomer of FAS-R ligand at the same time, thereby reducing or effectively neutralizing the TNF and FAS-R ligand activities at the cell surface in conditions, as noted above where excess amounts of these two cytokines are associated with undesirable cellular effects.
  • the FAS-R ligand is usually cell-surface-associated, and recent reports also describe cell-surface-associated forms of TNF.
  • these mixed TNF-R FAS-R oligomers are especially useful for neutralization of TNF and FAS-R ligand activities at the cell surface.
  • the present invention provides a DNA sequence encoding a protein capable of binding to one or more of the intracellular domains of one or more receptors belonging to the tumor necrosis factor/nerve growth factor (TNF/NGF) superfamily of receptors.
  • TNF/NGF tumor necrosis factor/nerve growth factor
  • the present invention provides a DNA sequence selected from the group consisting of :
  • the present invention also provides a DNA sequence selected from the group consisting of :
  • the DNA sequences encode p55 TNF-R, p75 TNF-R and FAS-R intracellular domain-binding proteins, such as those encoding the herein designated proteins 55.1, 55.3, 55.11, 75.3, 75.16, F2. F9 and DD11.
  • the present invention also provides a protein or analogs or derivatives thereof encoded by any of the above sequences of the invention, said proteins, analogs and derivatives being capable of binding to one or more of the intracellular domains of one or more TNF-Rs or FAS-R.
  • Embodiments of this aspect of the invention include the herein designated proteins 55.1, 55.3,
  • vectors encoding the above proteins of the invention which contain the above DNA sequences of the invention, these vectors being capable of being expressed in suitable eukaryotic or prokaryotic host cells; transformed eukaryotic or prokaryotic host cells containing such vectors, and a method for producing the proteins, analogs or derivatives of the invention by growing such transformed host cells under conditions suitable for the expression of said protein, analogs or derivatives, effecting post-translational modifications of said orotein as necessarv for ⁇ btention of said orotein and extractine said exoressed orotein analogs or derivatives from the culture medium of said transformed cells or from cell extracts of said transformed cells.
  • the present invention also provides antibodies or active derivatives or fragments thereof specific to the proteins, analogs and derivatives thereof, of the invention.
  • TNF-R or a FAS-R comprising treating said cells with one or more proteins, analogs or derivatives selected from the group consisting of the proteins, analogs and derivatives, according to the invention, and a protein being the p55IC, p55DD.
  • FAS-IC or FAS-DD analogs or derivatives thereof, all of said proteins being capable of binding to the intracellular domain and modulating the activity of said TNF-R or FAS-R, wherein said treating of the cells comprises introducing into said cells said one or more proteins.
  • R or a FAS-R comprising treating said cells with antibodies or active derivatives or fragments thereof according to the invention
  • R or FAS-R comprising treating said cells with an oligonucleotide sequence encoding an antisense sequence of at least part of the sequence according to the invention, or encoding an antisense sequence of the p55IC, p55DD. FAS-IC, or FAS-DD sequence. said oligonucleotide sequence being capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins;
  • R or FAS-R comprising :
  • a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a specific cell surface receptor and a sequence selected from an oligonucleotide sequence encoding an antisense sequence of at least part of the sequence according to the invention and an oligonucleotide sequence encoding an antisense sequence of the p55IC, p55DD, FAS-IC. or FAS-DD sequence, said oligonucleotide sequence being capable of blocking the expression of at least one of the TNF-R or FAS-R intracellular domain binding proteins when introduced into said cells by said virus ; and
  • R or a FAS-R comprising treating said cells with a suitable vector encoding a ribozyme having a sequence specific to a sequence selected from an mRNA sequence encoding a protein, analog or derivative of the invention and an mRNA sequence encoding the p55lC, p55DD, FAS-IC or FAS-DD, said ribozyme sequence capable of interacting with said mRNA sequence and capable of cleaving said mRNA sequence resulting in the inhibition of the expression of the protein, analog or derivative of the invention or of the expression of the p55IC, p55DD, FAS-IC or FAS-DD.
  • a recombinant animal virus vector carrying a sequence encoding a viral surface protein that is capable of binding to a tumor cell surface receptor or HTV- infected cell surface receptor or is capable of binding to another cell surface receptor of other diseased cells and a sequence selected from a sequence according to the invention encoding a protein, analog or derivative of the invention and a sequence encoding the p55IC, p55DD, FAS-IC, FAS-DD, or a biologically active analog or derivative thereof, said protein, analog or derivative of the invention, p55IC, p55DD, FAS-IC, FAS-DD, analog or derivative, when expressed in said tumor cell or HIV-infected cell, or other diseased cell being capable of killing said cell; and
  • a method for isolating and identifying proteins, factors or receptors capable of binding to the intracellular domain binding proteins according to the invention comprising applying the procedure of affinity chromatography in which said protein according to the invention is attached to the affinity chromatography matrix, said attached protein is brought into contact with a cell extract and proteins, factors or receptors from cell extract which bound to said attached protein are then eluted, isolated analyzed;
  • a method for isolating and identifying proteins, capable of binding to the intracellular domain binding proteins according to the invention comprising applying the yeast two-hybrid procedure in which a sequence encoding said intracellular domain binding protein is carried by one hybrid vector and a sequence from a cDNA or genomic DNA library is carried by the second hybrid vector, the vectors then being used to transform yeast host cells and the positive transformed cells being isolated, followed by extraction of the said second hybrid vector to obtain a sequence encoding a protein which binds to said intracellular domain binding protein; and (ix) a method for isolating and identifying
  • the present invention also provides a pharmaceutical composition for the modulation of the TNF- or FAS ligand- effect on cells comprising, as active ingredient, any one of the following : (i) a protein according to the invention, or the protein p55lC, p55DD.
  • FAS-IC or FAS-DD its biologically active fragments, analogs, derivatives or mixtures thereof;
  • a recombinant animal virus vector encoding a viral surface protein capable of binding to' a TNF-R or FAS-R - carrying cell - or tumor cell-specific receptor and a sequence encoding a protein, analog or derivative of the invention or encoding the p55IC, p55DD, FAS-IC or FAS-DD;
  • a specific embodiment of the above aspects of the invention is the use of the p55-IC or DNA encoding therefor
  • This embodiment is based on the discovery that the p55-IC may in a ligand (TNF)-independent fashion induce other TNF-associated effects in cells
  • a method for inducing TNF-associated effects in cells or tissues comprising treating said cells with one or more proteins, analogs or derivatives thereof, said one or more proteins being selected from a protein being essentially all of the self-associating intracellular domain of the p55 TNF-R (p55-lC) or portions thereof capable of self-associating and inducing, in a ligand (TNF)-independent manner, said TNF effect in the cells
  • said treating of the cells comprises introducing into said cells said one or more proteins, analogs or derivatives in a form suitable for intracellular introduction thereof, or introducing into said cells a DNA sequence encoding said one or more proteins, analogs or derivatives in the form of a suitable vector carrying
  • Embodiments of the above method of the invention include .
  • TNF effect to be induced in said cells is the induction of IL-8 gene expression, said vector carrying a sequence encoding essentially all of said p55-IC, portions thereof; analogs and derivatives of all of the foregoing, which are capable, when expressed in the cells of self-association and signaling for the induction of said IL-8 gene expression.
  • ( ⁇ i) a method for treating tumor cells or virally-infected cells, or for augmenting the antibacterial effect of granulocytes, wherein said viral vector carries a sequence encoding a viral ligand capable of binding a specific cell surface receptor on the surface of said tumor cells, virally- infected cells or granulocytes and a sequence encoding said p55-IC. po ⁇ ions thereof, analogs and derivatives thereof, which when expressed in said tumor, virally-infected or granulocyte cells induces TNF-associated effects leading to the death of these cells.
  • the intracellular domain of the p55-R (p55-IC), po ⁇ ions. analogs and derivatives of all of the aforegoing for use in the treatment of cells by induction therein of TNF-associated effects; and the following embodiments thereo : i) the p55-IC, portions, analogs and derivatives for use in the treatment of cells by induction therein pf IL-8 gene expression.
  • a pharmaceutical composition for treating cells by induction therein of TNF-associated effects comprising, as active ingredient, p55-IC, portions thereof, analogs and derivatives of all of the aforegoing, and a pharmaceutically acceptable carrier; and the following embodiments thereof :
  • a pharmaceutical composition for treating cells by induction therein of TNF-associated effects comprising, as active ingredient a recombinant animal virus vector encoding p55-IC, portions thereof, analogs and derivatives of all of the aforegoing, and a protein capable of binding a cell surface protein on the cells to be treated.
  • the present invention provides a soluble, oligomeric tumor necrosis factor receptor (TNF-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a TNF binding domain selected from the extracellular domain of a TNF-R, analogs or derivatives thereof, said extracellular domain, analogs or derivatives thereof being incapable of deleterious self-association and being able to bind TNF; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R ( ⁇ 55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-R), (ii) the death domain of the p55-IC extending from about amino acid residue 328 to about amino acid residue 426 of the native p55-R; (iii) essentially all of the intracellular domain of the Fas/APOl receptor (F
  • Embodiments of this aspect of the invention include all of the above combinations of ends (a) with ends(b) as defined above, for example, a soluble, oligomeric TNF-R comprising as extracellular domain, the p55-R extracellular domain and as self-associating intracellular domain, the ⁇ 55-IC.
  • a vector encoding the above fusion proteins, useful in the above method of the invention; host cells containing the vector; as well as a pharmaceutical composition comprising the soluble, oligomeric TNF-R, salts or functional derivatives thereof and mixtures of any of the aforegoing according to the invention, as active ingredient, together with a pharmaceutically acceptable carrier.
  • the soluble, oligomeric TNF-R, salts, functional derivatives thereof and mixtures of any of the aforegoing, according to the invention are provided for use in antagonizing the deleterious effect of TNF in mammals, especially in the treatment of conditions wherein an excess of TNF is formed endogenously or is exogenously administered; or alternatively, for use in maintaining prolonged beneficial effects of TNF in mammals when used with TNF exogenously administered.
  • the present invention provides a soluble, oligomeric Fas/APOl receptor (Fas-R) comprising at least two self-associated fusion proteins, each fusion protein having (a) at its one end, a Fas ligand binding domain selected from the extracellular domain of a Fas-R, analogs or derivatives thereof being incapable of self-associating and being able to bind Fas ligand; and (b) at its other end, a self-associating domain selected from (i) essentially all of the intracellular domain of the p55 TNF-R (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-IC), extending from about amino acid residue 206 to about amino acid residue 426 of the native p55 TNF-R molecule (p55-IC), extending from
  • an expression vector containing the fusion protein sequence encoding the soluble oligomeric Fas-R useful in the above process; host cells containing the vector; and pharmaceutical compositions comprising the soluble, oligomeric Fas-R, salts or functional derivatives thereof or mixtures of any of the aforegoing as active ingredient together with a pharmaceutically acceptable carrier.
  • a soluble, oligomeric Fas- R, salts or functional derivatives thereof or mixtures of any of the aforegoing for use in antagonizing the deleterious effect of Fas ligand in mammals, especially in the treatment of conditions wherein an excess of the Fas ligand is formed endogenously or is exogenously administered.
  • the present invention also provides a mixed oligomeric TNF-R/FAS-R comprising at least two self-associated fusion proteins, one of which fusion proteins is selected from any one of the above mentioned TNF-specific fusion proteins, and the other fusion protein is selected from any one of the above mentioned FAS-R ligand-specific fusion proteins, to provide a mixed oligomer having at least one TNF-R extracellular domain and at least one FAS-R extracellular domain associated by virtue of the self-association between the intracellular domains or portions thereof fused to each of these extracellular domains.
  • These mixed oligomeric receptors are prepared by preparing, as noted above, the oligomeric TNF-Rs and the oligomeric FAS-Rs and then mixing these together and subsequently selecting, by standard procedures, those oligomers having binding specificity for both FAS-R ligand and TNF.
  • Another way for preparing the mixed oligomeric receptors is by co-transfecting suitable host cells with vectors, as noted above, encoding any of the TNF-specific fusion proteins (soluble TNF-Rs) and encoding any of the FAS-R ligand-specific fusion proteins (soluble FAS-Rs), purifying the expressed fusion proteins which self-associate prior to, during, or following the purification to yield oligomeric receptors, and then selecting by standard procedures, those oligomeric receptors which are capable of binding to both TNF and FAS-R ligand.
  • compositions comprising the mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the aforegoing as active ingredient together with a pharmaceutically acceptable carrier.
  • the mixed oligomeric receptors, salts or functional derivatives thereof or mixtures of any of the aforegoing for use in antagonizing the deleterious effects of both TNF and FAS-R ligand in mammals, especially in the treatment of conditions wherein an excess of TNF and FAS-R ligand is formed endogenously or is exogenously administered; or alternatively, for use in maintaining prolonged (slow-release) beneficial effects of TNF and/or FAS-R ligand in mammals when used with TNF and/or FAS-R ligand (in soluble form) exogenously administered.
  • Other aspects and embodiments of the present invention are also provided as arising from the following detailed description of the invention.
  • Figs la-c depict schematically the partial and preliminary nucieotide sequence of cDNA clones encoding the p55IC and p75lC-binding proteins
  • Fig. 1(a) is the sequence of clone 55.11 encoding the p55IC-binding protein 55.11
  • Fig. 1(b) is the partial and preliminary sequence of clone 75.3 encoding the p75IC-binding protein 75.3
  • Fig 1(c) is the partial and preliminary sequence of clone 75.16 encoding the p751C-binding protein p75.16; all as described in Example 1
  • Fig. 1(d) depicts the deduced amino acid sequence of protein 55.11, deduced from the nucieotide sequence of Fig. 1(a), as also described in Example 1.
  • Fig. 2 is a reproduction of a Northern blot which shows the 55.11 -specific mRNAs present in a number of tested cell lines, as described in Example 1.
  • Figs. 3A and B are reproductions of autoradiograms depicting the in vitro binding of the protein encoded for by the 55.11 cDNA to GST fusion proteins containing portions of p55-IC, wherein in Fig. 3A there is depicted the binding of the full-length 55.1 1 protein (55.11 full to the various GST fusion proteins; and in Fig. 3B there is depicted the binding of a portion of 55.11 fused to the FLAG octapeptide to the various GST fusion proteins, all as described in Example 1 , Fig. 4 shows schematically a comparison of the deduced amino acid sequence of human 55.11 to related protein sequences derived from lower organisms, as described in Example 1. Fig.
  • FIG. 5 is a reproduction of a Western blot stained with anti-MBP poiyclonal antiserum. showing the self association of the p55IC, the Western blot derived from an SDS-PAGE gel on which were electrophoresed the interacting bacterially-produced chimeric proteins p55IC- MBP and p55IC-GST (lanes 1-4) or the control interaction between the chimeric protein p55IC-MBP and GST alone (lanes 5-8), the interactions between the chimeric proteins (and control) being carried out on glutathion-agarose beads prior to SDS-PAGE, as described in Example 2.
  • FIG. 6 is a reproduction of phase contrast micrographs showing the cytotoxic effect of the full- length p55IC in HTtal cells transfected with an expression vector encoding this p55IC (right panel); and the inhibition of this cytotoxic effect when expression of the vector is blocked by treating the cells with tetracycline (left panel), as described in Example 2.
  • Fig. 7 depicts the ligand-independent triggering of the cytocidal effect in HeLa cells transfected with the full-length p55-R, its intracellular domain, or parts of the intracellular domain including the 'death domain * where :
  • the left and middle bar graphs show the TNF receptor expression in the HeLa cells of each of the types of receptor shown at the extreme left of Fig. 7, the left bar representing the amounts of receptor in ng/cell sample and the mid le bar ⁇ p representing the amounts of receptor expressed in terms of radioiodinated TNF bound to the transfected cells;
  • Fig. 8 depicts the ligand-independent induction of IL-8 gene expression in HeLa cells transfected with the full-length p55-R or its intracellular domain (p55lC), wherein in panel A there is shown a reproduction of a Northern blot representing the Northern analysis of RNA extracted from HeLa cells treated or untreated with TNF (two left hand lanes marked 'control' and TNF'), and of RNA extracted from HeLa cells transfected with vectors encoding the p55-R, p55-IC or the control protein, iuciferase (the remaining lanes marked 'p55-IC', 'p55-R' and Luc, respectively), the cells having been transfected in the presence (+) or absence (-) of tetracycline in each case (hence two lanes per transfection); and wherein in panel B there is shown the methylene biue staining of 18S rRNA in each of the
  • Fig. 9 depicts graphically the ligand independent triggering of a cytocidal effect in
  • Fig. 9A there is depicted the results with respect to the p55R or pans thereof and in Fig. 9B there is depicted the results with respect to the FAS-IC.
  • Fig. 9B there is depicted the results with respect to the FAS-IC.
  • Fig. 10 depicts schematically the partial and preliminary nucieotide sequence of a cDNA clone, called ⁇ z2 which encodes a protein capable of binding to the p55IC and FAS-IC, as described in Example 3.
  • Fig. 11 depicts schematically the partial and preliminary nucieotide sequence of a cDNA clone, called F9, which encodes a protein capable of binding to the p55IC and FAS-IC, as described in Example 3
  • Fig. 12 depicts schematically the partial and preliminary nucieotide sequence of a cDNA clone, called DDl l . which encodes a protein capable of binding to the p55IC, especially the p55DD, and FAS-IC, as described in Example 3
  • the present invention relates, in one aspect, to novel proteins which are capable of binding to the intracellular domain of receptors belonging to the TNF NGF superfamily, such as TNF-Rs and FAS-R and hence are considered as mediators or modulators of this superfamily of receptors, e.g. of the TNF-Rs and FAS-R, having a role in, for example, the signaling process that is initiated by the binding of TNF to the TNF-R and FAS ligand to FAS-R.
  • these proteins are those which bind to the intracellular domain of the p55 TNF-R (p55IC), such as the proteins designated herein as 55.1, 55.3 and 55.11 (Example 1) as well as those encoded by cDNA clones
  • Proteins 55.1 and 55.3 have been found to represent portions or fragments of the intracellular domain of the p55 TNF-R (p55IC); other proteins,
  • the new proteins encoded by cDNA clones F2, F9 and DDl l also represent proteins previously not described at all, i.e. their sequence is not in the 'GENEBANK' or
  • PROTEIN BANK' data banks of DNA or amino acid sequences.
  • the present invention concerns the DNA sequences encoding these proteins and the proteins encoded by these sequences. Moreover, the present invention also concerns the DNA sequences encoding biologically active analogs and derivatives of these proteins, and the analogs and derivatives encoded thereby.
  • the preparation of such analogs and derivatives is by standard procedure (see for example, Sambrook et al., 1989) in which in the DNA sequences encoding these proteins, one or more codons may be deleted, added or substituted by another, to yield analogs having at least a one amino acid residue change with respect to the native protein.
  • Acceptable analogs are those which retain at least the capability of binding to the intracellular domain of the TNF/NGF receptor superfamily, such as FAS-R or TNF-R, e.g.
  • analogs which bind the p55, p75IC or FAS-IC but which do not signal i e. do not bind to a further downstream receptor, protein or other factor, or do not catalyze a signal-dependent reaction.
  • analogs can be produced which have a so-called dominant-negative effect, namely, an analog which is defective either in binding to the, for example, p55IC, p75IC or FAS-IC, or in subsequent signaling following such binding.
  • Such analogs can be used, for example, to inhibit the TNF- or FAS-ligand- effect by competing with the natural IC-binding proteins.
  • so-called dominant-positive analogs may be produced which would serve to enhance, for example, the TNF or FAS ligand effect. These would have the same or better IC-binding properties and the same or better signaling properties of the natural IC- binding proteins.
  • derivatives may be prepared by standard modifications of the side groups of one or more amino acid residues of the proteins, or by conjugation of the proteins to another molecule e.g an antibody, enzyme, receptor, etc., as are well known in the art.
  • the new- TNF-R and FAS-R intracellular domain - binding proteins e.g. the proteins 55. . ,
  • F2. F9 and DDll have a number of possible uses, for example:
  • TNF or FAS-R ligand may be used to mimic or enhance the function of TNF or FAS-R ligand, in situations where an enhanced TNF or FAS-R ligand effect is desired such as in anti-t mcr, anti-inflammatory or anti-HTV applications where the TNF-or FAS-R ligand- induced cytotoxicity is desired.
  • the proteins e.g. those binding to the p55IC such as
  • proteins F2, F9 and DDll as well as FAS-IC and FAS-DD which enhance the FAS-R ligand effect, i.e. cytotoxic effect, may be introduced to the cells by standard procedures known p_er se.
  • proteins are intracellular and it is desired that they be introduced only into the cells where the TNF or FAS-R ligand effect is wanted, a system for specific introduction of these proteins into the cells is necessary.
  • One way of doing this is by creating a recombinant animal virus e.g.
  • the gene encoding a ligand that binds to cell surface proteins specifically expressed by the cells e.g. ones such as the AIDs (HIV) virus gpl20 protein which binds specifically to some cells (CD4 lymphocytes and related leukemias) or any other ligand that binds specifically to cells carrying a TNF-R or FAS-R, such that the recombinant virus vector will be capable of binding such TNF-R- or FAS-R- carrying cells; and the gene encoding the new intracellular domain-binding protein or the p55IC, p55DD, FAS-IC or FAS-DD protein.
  • expression of the cell-surface-binding protein on the surface of the virus will target the virus specifically to the tumor cell or other TNF-R- or FAS-R- carrying cell, following which the intracellular domain-binding protein encoding sequence or p55IC, p55DD, FAS-IC or FAS-DD encoding sequence will be introduced into the cells via the virus, and once expressed in the cells will result in enhancement of the TNF or FAS-R ligand effect leading to the death of the tumor cells or other TNF-R- or FAS-R- carrying cells it is desired to kill.
  • Construction of such recombinant animal virus is by standard procedures (see for example, Sambrook et al., 1989).
  • Another possibility is to introduce the sequences of the new proteins or the p55IC, p55DD, FAS-IC or FAS-DD in the form of oligonucleotides which can be absorbed by the cells and expressed therein.
  • TNF or FAS-R ligand effect may be used to inhibit the TNF or FAS-R ligand effect, e.g. in cases such as tissue damage in septic shock, graft-vs.-host rejection, or acute hepatitis, in which case it is desired to block the TNF-induced TNF-R. or FAS-R ligand induced FAS-R intracellular signaling.
  • Such oligonucleotides may be introduced into the cells using the above recombinant virus approach, the second sequence carried by the virus being the oligonucleotide sequence.
  • Another possibility is to use antibodies specific for these proteins to inhibit their intracellular signaling activity. It is possible *hat these new proteins have an extracellular domain as well as an intracellular one, the latter which binds to the TNF-R or FAS-R binding domain, and thus antibodies generated to their extracellular domains can be used to block their TNF- or FAS-R ligand- related functions.
  • Ribozymes are catalytic RNA molecules that specifically cleave RNAs. Ribozymes may be engineered to cleave target RNAs of choice, e.g. the mRNAs encoding the new proteins of the invention or the mRNA encoding the p55IC, p55DD, FAS-IC or FAS-DD.
  • ribozymes would have a sequence specific for the mRNA of choice and would be capable of interacting therewith (complementary binding) followed by cleavage of the mRNA, resulting in a decrease (or complete loss) in the expression of the protein it is desired to inhibit, the level of decreased expression being dependent upon the level of ribozyme expression in the target cell.
  • any suitable vector may be used, e.g. plasmid, animal virus (retrovirus) vectors, that are usually used for this purpose (see also (i) above, where the virus has, as second sequence, a cDNA encoding the ribozyme sequence of choice).
  • ribozymes can be constructed which have multiple targets (multi-target ribozymes) that can be used, for example, to inhibit the expression of one or more of the proteins of the invention and/or the p55IC, p55DD, FAS-IC or FAS-DD as well (For reviews, methods etc. concerning ribozymes see Chen et al., 1992; Zhao and Pick, 1993, Shore et al., 1993. Joseph and Burke, 1993,
  • the yeast two-hybrid system it may be specifically tested whether the proteins of the present invention are capable of specifically binding to these other TNF-Rs or other receptors of the TNF/NGF superfamily. Moreover, this approach may also be taken to determine whether the proteins of the present invention are capable of binding to other known receptors in whose activity they may have a functional role. (iv) The new proteins may also be used to isolate, identify and clone other proteins of the same class i.e.
  • yeast two-hybrid system may be used, or there may be used a recently developed (Wilks et al virgin 1989) system employing non-stringent southern hybridization followed by PCR cloning.
  • Wilks et al. publication there is described the identification and cloning of two putative protein-tyrosine kinases by application of non-stringent southern hybridization followed by cloning by PCR based on the known sequence of the kinase motif, a conceived kinase sequence. This approach may be used, in accordance with the present invention using the sequences of the new proteins to identify and clone those of related TNF-R, FAS-R or related receptor (TNF/NGF superfamily receptors) intracellular domain-binding proteins.
  • proteins of the present invention may be individually attached to affinity chromatography matrices and ther. brought into contact with cell extracts or isolated proteins or factors suspected of being involved in the intracellular signaling process.
  • the new proteins of the invention may also be used as immunogens (antigens) to produce specific antibodies thereto. These antibodies may also be used for the purposes of purification of the new proteins either from cell extracts or from transformed cell lines producing them. Further, these antibodies may be used for diagnostic purposes for identifying disorders related to abnormal functioning of the TNF or FAS-R ligand system, e.g overactive or underactive TNF- or FAS-R ligand- induced cellular effects Thus, should such disorders be related to a malfunctioning intracellular signaling system involving the new proteins, such antibodies would serve as an important diagnostic tool.
  • the isolation, identification and characterization of the new proteins of the invention may be performed using any of the well known standard screening procedures. For example, one of these screening procedures, the yeast two-hybrid procedure as is set forth in the following examples (Examples 1 and 3), was used to identify the new proteins of the invention. Likewise as noted above and below, other procedures may be employed such as affinity chromatography, DNA hybridization procedures, etc. as are well known in the art, to isolate, identify and characterize the new proteins of the invention or to isolate, identify' and characterize additional proteins, factors, receptors, etc. which are capable of binding to the new proteins of the invention or to the receptors belonging to the TNF/NGF family of re eptors.
  • antibody is meant to include polyclonal antibodies, monoclonal antibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled in soluble or bound form, as well as fragments thereof provided by any known technique, such as. but not limited to enzymatic cleavage, peptide synthesis or recombinant techniques.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • a monoclonal antibody contains a substantially homogeneous population of antibodies specific to antigens, which populations contains substantially similar epitope binding sites MAbs may be obtained by methods known to those skilled in the art. See. for example Kohler and Milstein, Nature. 256:495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds., Harlow and Lane ANTIBODIES • A LABORATORY MANUAL, Cold Spring Harbor Laboratory (1988); and Colligan et al., eds.. Current Protocols in Immunology. Greene publishing Assoc.
  • Such antibodies may be of any immunoglobulin class including IgG. IgM, IgE, IgA, GILD and any subclass thereof.
  • a hybridoma producing a mAb of the present invention may be cultivated in virro, in situ or w vivo. Production of high titers of mAbs in vivo or in situ makes this the presently preferred method of production.
  • Chimeric antibodies are molecules different portions of which are derived from different animal species, such as those having the variable region derived from a murine mAb and a human immunoglobulin constant region. Chimeric antibodies are primarily used to reduce immunogenicity in application and to increase yields in production, for example, where murine mAbs have higher yields from hybridomas but higher immunogenicity in humans, such that human/murine chimeric mAbs are used. Chimeric antibodies and methods for their production are known in the art (Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci.
  • An anti-idiotypic (anti-Id) antibody is an antibody which recognizes unique determinants generally associated with the antigen-binding site of an antibody.
  • An Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the mAb with the mAb to which an anti-Id is being prepared. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody by producing an antibody to these idiotypic determinants (the anti-Id antibody). See, for example. U.S. Patent No. 4,699,880, which is herein entirely incorporated by reference.
  • the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-anti-Id antibody.
  • the anti-anti-Id may be epitopically identical to the original mAb which induced the anti-Id.
  • antibodies to the idiotypic determinants of a mAb it is possible to identify other clones expressing antibodies of identical specificity.
  • mAbs generated against the IC-binding proteins, analogs or derivatives thereof, of the present invention or the p55IC, p55DD, FAS-IC, FAS-DD, analogs or derivatives thereof may be used to induce anti-Id antibodies in suitable animals, such as BALB/c mice.
  • Spleen cells from such immunized mice are used to produce anti-Id hybridomas secreting anti-Id mAbs.
  • the anti-Id mAbs can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize additional BALB/c mice.
  • KLH keyhole limpet hemocyanin
  • Sera from these mice will contain anti-anti-Id antibodies that have the binding properties of the original mAb specific for an epitope of the above IC-binding proteins, analogs or derivatives or p55IC.
  • anti-Id mAbs thus have their own idiotypic epitopes, or "idiotopes" structurally similar to the epitope being evaluated, such as GRB protein- ⁇ .
  • antibody is also meant to include both intact molecules as well as fragments thereof, such as, for example, Fab and F(ab')2. which are capable of binding antigen.
  • Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)).
  • Fab and F(ab')2 and other fragments of the antibodies useful in the present invention may be used for the detection and quantitation of the IC-binding proteins or p55IC, p55DD.
  • FAS-IC or FAS-DD according to the methods disclosed herein for intact antibody molecules.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • An antibody is said to be "capable of binding" a molecule if it is capable of specifically reacting with the molecule to thereby bind the molecule to the antibody.
  • epitope is meant to refer to that portion of any molecule capable of being bound by an antibody which can also be recognized by that antibody.
  • Epitopes or “antigenic determinants” usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics
  • an "antigen” is a molecule or a portion of a molecule capable of being bound by an antibody which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen may have one or more than one epitope. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of o'her antibodies which may be evoked by other antigens.
  • the antibodies, including fragments of antibodies, useful in the present invention may be used to quantitatively or qualitatively detect the IC-binding proteins or p55IC.
  • p55DD, FAS-IC, FAS-DD in a sample or to detect presence of cells which express the IC-binding proteins of the present invention or the p55IC, p55DD, FAS-IC, FAS-DD proteins.
  • This can be accomplished by immunofluorescence techniques employing a fl ⁇ orescently labeled antibody (see below) coupled with light microscopic, flow cytometric, or fluorometric detection.
  • the antibodies (or fragments thereof) useful in the present invention may be employed histologically, as in immunofluorescence or immunoelectron microscopy, for in situ detection of IC-binding proteins of the present invention or the p55IC, p55DD, FAS-IC. FAS-DD.
  • In situ detection may be accomplished by removing a histological specimen from a patient, and providing the labeled antibody of the present invention to such a specimen.
  • the antibody (or fragment) is preferably provided by applying or by overlaying the labeled antibody (or fragment) to a biological sample.
  • Such assays for IC-binding proteins of the present invention or the p55IC, p55DD, FAS- IC, FAS-DD typically comprises incubating a biological sample, such as a biological fluid, a tissue extract, freshly harvested cells such as lymphocytes or leukocytes, or cells which have been incubated in tissue culture, in the presence of a detectably labeled antibody capably of identifying the IC-binding proteins or the p55IC, p55DD, FAS-IC. FAS-DD. and detecting the antibody by any of a number of techniques well known in the art.
  • the biological sample may be treated with a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins.
  • a solid phase support or carrier such as nitrocellulose, or other solid support or carrier which is capable of immobilizing cells, cell particles or soluble proteins.
  • the support or carrier may then be washed with suitable buffers followed by treatment with a detectably labeled antibody in accordance with the present invention, as noted above.
  • the solid phase support or carrier may then be washed with the buffer a second time to remove unbound antibody.
  • the amount of bound label on said solid support or carrier may then be detected by conventional means.
  • solid phase support By “solid phase support”, “solid phase carrier”, “soiid support”, “solid carrier”, “support” or “carrier” is intended any support or carrier capable of binding antigen or antibodies
  • supports or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases. natural and modified celluloses, polyacrylamides. gabbros and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention.
  • the support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to an antigen or antibody.
  • the support or carrier configuration may be spherical, as in a bead, cylindrical, as in the inside surface of a test tube, or the external surface of a rod.
  • the surface may be flat such as a sheet, test strip, etc.
  • Preferred supports or carriers include polystyrene beads. Those skilled in the art will know may other suitable carriers for binding antibody or antigen, or will be able to ascertain the same by use of routine experimentation.
  • binding activity of a given lot of antibody, of the invention as noted above may be determined according to well known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.
  • an antibody in accordance with the present invention can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA), This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means.
  • EIA enzyme immunoassay
  • Enzymes which can be used detectably label the antibody include, but are not limited to, malate dehydrogenase.
  • staphylococcal nuclease delta- 5-steroid isomeras
  • yeast alcohol dehydrogenase alpha-glycerophosphate dehydrogenase
  • triose phosphate isomerase horseradish peroxidase, alkaline phosphatase, asparaginase. glucose oxidase. beta-galactosidase, ribonuciease, urease, cataiase. glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the detection can be accomplished by colorimetric methods which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.
  • Detection may be accomplished using any of a variety of other immunoassays.
  • a radioimmunoassay RIA
  • a good description of RIA may be found in Laboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company. NY (197S) with particular reference to the chapter entitled "An Introduction to Radioimmune Assay and Related Techniques" by Chard, T., incorporated by reference herein,
  • the radioactive isotope can be detected by such means as the use of a ⁇ counter or a scintillation counter or by autoradiography.
  • an antibody in accordance with the present invention with a fluorescent compound.
  • fluorescent labeling compounds are fluorescein isothiccyanate, rhodamine, phycoerythrine, pycocyanin, allophycocya ⁇ in, o-phthaldehyde and fluorescamine.
  • the antibody can also be detectably labeled using fluorescence emitting metals such as ⁇ 2 E, or others of the la ⁇ thanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).
  • fluorescence emitting metals such as ⁇ 2 E, or others of the la ⁇ thanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriamine pentaacetic acid (ETPA).
  • the antibody can also be detectably labeled by coupling it to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are iuminol, isoluminol. theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence. Important bioluminescent compounds for purposes of labeling are lu ⁇ ferin, luciferase and aequorin.
  • An antibody molecule of the present invention may be adapted for utilization in an immunometric assay, also known as a "two-site” or “sandwich” assay.
  • an immunometric assay also known as a "two-site” or “sandwich” assay.
  • a quantity of unlabeled antibody (or fragment of antibody) is bound to a solid support or carrier and a quantity of detectably labeled soluble antibody is added to permit detection and/or quan ⁇ tation of the ternary complex formed between solid-phase antibody, antigen, and labeled antibody.
  • Typical, and preferred, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample being tested to extract the antigen from the sample by formation of a binary solid phase antibody-antigen complex.
  • the solid support or carrier is w-ashed to remove the residue of the fluid sample, including unreacted antigen, if any, and the contacted with the solution containing an unknown quantity of labeled antibody (which functions as a "reporter molecule").
  • the solid support or carrier is washed a second time to remove the unreacted labeled antibody.
  • the so-called “simultaneous” and “reverse” assays are used.
  • a simultaneous assay involves a single incubation step as the antibody bound to the solid support or carrier and labeled antibody are both added to the sample being tested at the same time. After the incubation is completed, the solid support or carrier is washed to remove the residue of fluid sample and uncomplexed labeled antibody. The presence of labeled antibody associated with the solid support or carrier is then determined as it would be in a conventional "forward" sandwich assay.
  • stepwise addition first of a solution of labeled antibody to the fluid sample followed by the addition of unlabeled antibody bound to a solid support or carrier after a suitable incubation period is utilized. After a second incubation, the solid phase is washed in conventional fashion to free it of the residue of the sample being tested and the solution of unreacted labeled antibody. The determination of labeled antibody associated with a solid support or carrier is then determined as in the "simultaneous" and "forward" assays.
  • the new proteins of the invention once isolated, identified and characterized by any of the standard screening procedures, for example, the yeast two-hybrid method, affinity chromatography, and any other well known method known in the art, may then be produced hy any standard recombinant DNA procedure (see for example, Sambrook, et al, 1989) in which suitable eukaryotic or prokaryotic host cells are transformed by appropriate eukaryotic or prokaryotic vectors containing the sequences encoding for the proteins. Accordingly, the present invention also concerns such expression vectors and transformed hosts for the production of the proteins of the invention.
  • these proteins also include their biologically active analogs and derivatives, and thus the vectors encoding them also include vectors encoding analogs of these proteins, and the transformed hosts include those producing such analogs.
  • the derivatives of these proteins are the derivatives produced by standard modification of the proteins or their analogs, produced by the transformed hosts.
  • the invention relates to the use of the free intracellular domain of the ⁇ 55 TNR-R (p55IC) or FAS-R (FAS-IC) or their so-called 'death domains' (p55DD or FAS-DD, respectively) as an agent for enhancing the TNF or FAS-R ligand effect on cells, on its own (see Example 2).
  • the p55IC, p55DD, FAS-IC or FAS-DD can be introduced into such cells using the above noted (see (i) above) recombinant animal virus (e.g. vaccinia) approach.
  • the native ⁇ 55IC. p55DD. AS-IC or FAS-DD, biologically active analogs and derivatives or fragments may be used, all of which can be prepared as noted above.
  • the present invention also relates to the specific blocking of the TNF-effect or FAS-R ligand-effect by blocking the activity of the p55IC, p55DD, FAS-IC or FAS-DD, e.g. anti- sense oligonucleotides may be introduced into the cells to block the expression of the p55IC , p55DD, FAS-IC or FAS-DD.
  • the present invention also relates to pharmaceutical compositions comprising recombinant animal virus vectors encoding the TNF-R or FAS-R intracellular domain binding proteins
  • virus surface protein capable of binding specific target cell (e.g. cancer cells) surface proteins to direct the insertion of the intracellular domain binding protein sequences into the cells.
  • the present invention also concerns, specifically, the effects of the self- associating intracellular domain of the p55 TNF receptor (p55-IC. see Example 2).
  • p55-IC p55 TNF receptor
  • IL-8 is a cytokine belonging to the subclass of chemokines having primarily chemotactic activity, and has been shown to play a major role in the chemotaxis of granulocytes and other cell types associated with a number of pathological states (see for example, Endo et al., 1994; Sekido et al., 1993; Harada et al., 1993; Ferrick et al., 1991).
  • TNF has a beneficial activity, and is used as such, in treatments to destroy tumor cells and virus infected cells or to augment antibacterial activities of granulocytes.
  • TNF also has undesirable activities in which case it is desired to block its activity, including those situations where large doses of TNF are used in cancer therapy, antiviral therapy or antibacterial therapy.
  • TNF tumor necrosis factor
  • a substance capable of mimicking s beneficial activity to the cells or tissues that it is specifically desired to treat.
  • the self-associating intracellular domain of the p55-R can, in a ligand-independent manner, mimic a number of effects of TNF, e.g. the 'death domain' of p55-IC can induce cytotoxic effects on cells, and that the p55-IC can induce IL-8 gene expression.
  • the p55-IC can mimic TNF function in a site-directed fashion, i.e. to introduce the ⁇ 55-IC only to those cells or tissues it is desired to treat.
  • One example of the above approach is to specifically transfect (transform) tumor cells or malignant tissue with a DNA molecule encoding p55-IC or a po ⁇ ion thereof which can induce not only cytotoxic effects on such cells or tissue but also augment these effects by the co-induction of IL-8, which will result in the accumulation at the site of these cells or tissue of granulocytes and other lymphocytes, which, in turn, will serve to destroy the tumor cells or tissue.
  • This approach obviates the need for administration of large doses of TNF with its associated deleterious side-effects
  • it is possible to prepare various regions of the p55-IC and to determine which region is responsible for each TNF-induced effect e.g.
  • the 'death domain' is responsible for cytotoxicity (Example 2), and we have already prepared various other constructs containing po ⁇ ions of the p55-IC, which po ⁇ ions (together with part or all of the death domain) may be responsible for other TNF-effects, and which may be used in a ligand-independent manner, once self-associated for activity, to induce these effects, e.g. IL-8 induction.
  • sequence of the p55-IC involved in the induction of other TNF- associated effects may be different to that involved in cytotoxicity, i.e. may include none or only part of the 'death domain' and have other sequence motifs from other regions of the intracellular domain, or may be the same sequence, different features of the sequence (same sequence motif) being involved in the induction of different effects
  • expression vectors containing these p55-IC portions, analogs or derivatives thereof may be prepared, expressed in host cells, purified and tested for their activity.
  • a number of such p55-IC fragments having one or more TNF- associated activities may be prepared and used in a differential fashion for the treatment of any number of pathological conditions, e.g. viral infections, bacterial infections, tumors, etc.
  • the specific activity can be augmented by incorporation (or co-transfection) with the p55-IC fragment responsible for IL-8 gene expression induction, permitting the desirable IL-8 chemotactic activity to enhance the destruction of the cells or tissues it is desired to destroy.
  • the p55-IC may be introduced specifically into the cells or tissues it is wished to destroy by any one of the abovementioned procedures.
  • a recombinant animal virus e.g. one derived from Vaccinia, to whose DNA the following two genes will be introduced : the gene encoding a ligand that binds to cell surface proteins specifically expressed by the cells e.g. ones such as the AIDS virus gpl20 protein which binds specifically to some cells (CD4 lymphocytes and related leukemias) or any other ligand that binds specifically to cells carrying a TNF-R.
  • the recombinant virus vector will be capable of binding such TNF-R-carrying cells; and the gene encoding the p,55-lC or a portion thereof.
  • expression of the cell-surface-binding protein on the surface of the virus will target the virus specifically to the tumor cell or other TNF-R-carrying cell, following which the p55-IC, or portion thereof, encoding sequence will be introduced into the cells via the virus, and once expressed in the cells will result in enhancement of the TNF effect leading to the death of the tumor cells or other TNF-R-carrying cells it is desired to kill or induction, for example, of IL-8 which will lead to cell death.
  • compositions comprising the above recombinant animal virus vectors encoding the p55-IC or portions thereof, which vector also encodes a virus surface protein capable of binding specific target cell (e.g. cancer cells) surface proteins to direct the insertion of the p55-lC, or portions thereof, sequence into the cells.
  • target cell e.g. cancer cells
  • the present invention relates, in yet another aspect, to new synthetic TNF receptors which are soluble and capable of oligomerization to form dimeric, and possibly also high order multimeric, TNF receptor molecules, each monomeric part of these receptors being capable of binding to a TNF monomer, TNF occurs naturally as a homotrimer containing three, active TNF monomers, each capable of binding to a single TNF receptor molecule, while TNF receptors occur naturally as monomers each capable of binding only one of the monomers of the TNF homotrimeric molecule.
  • TNF when TNF binds to TNF receptors on the cell surface, it is capable of binding to three receptor molecules resulting in the clustering of the TNF receptors, which is believed to be the start of the signaling process which ultimately triggers the observed TNF effects on the cells.
  • TNF has many desirable effects such as its ability to destroy, for example, tumor cells or virus-infected cells and to augment antibacterial activities of granulocytes
  • TNF does however, have many undesirable effects such as, for example, in many severe diseases including autoimmune disorders, rheumatoid arthritis, graft-versus-host reaction (graft rejection), septic shock, TNF has been implicated as the major cause for pathological tissue destruction.
  • TNF may also cause excessive loss of weight (cachexia) by suppressing the activities of adipocytes.
  • cachexia excessive loss of weight
  • TNF-blocking agents including soluble proteins capable of binding TNF and inhibiting its binding to its receptors and hence also inhibiting the cytotoxic effects of TNF (see EP 308378, EP 398327 and EP 568925).
  • these TNF binding proteins, or soluble TNF receptors are monomeric. each binding only one of the TNF monomers of the TNF homotrimer. Hence, the blocking of the TNF function may not be complete, each monomeric receptor-bound TNF molecule still having two TNF monomers free to be able to bind cell-surface TNF receptors and illicit its effects on the cells.
  • soluble oligomeric TNF receptors which are capable of binding at least two TNF monomers of the naturally occurring TNF homotrimer molecule.
  • these soluble oligomeric TNF receptors bind more avidly to their TNF ligand than the previously known monomeric soluble TNF binding proteins or receptors.
  • the soluble TNF receptor of the invention is in the form of a dimer, it is capable of binding two TNF monomers of a TNF trimer and hence causer 2 mor? coms.et? neutralizaticr. of the TNF.
  • the basis for the development of the soluble oligomeric TNF receptors of the invention was the discovery that the intracellular domain of the p55-R TNF receptor was capable of self- association, and further, that within this intracellular domain (p55-lC) there exists a region, the so-called 'death domain', which is also capable of self-association and as such, in a ligand- independent fashion, can cause cytotoxic effects cn cells (see Example 2).
  • a fusion protein Utilizing this self- association property of the p55-IC and its 'death domain' it is thus possible to construct a fusion protein, using standard recombinant DNA technology, containing essentially all of the extracellular domain of a TNF receptor such as the p75-R or p55-R receptors, preferably the p55- R, and fused thereto, essentially all of the intracellular domain (p55-IC) or the death domain of the p55-IC.
  • a new fusion product is produced which has at one end the TNF binding domain i.e., the extracellular domain of the receptor, and at its other end the intracellular domain or the death domain thereof which is capable of self-association. Accordingly, such a product can oligomerize by self-association between two (and possible more) p55-lC or death domains thereof to yield oiigomers (or at least di ers) having at least two TNF binding domains.
  • the Fas/APOl receptor has a self-associating, intracellular domain inclusive of a self-associatmg 'death domain' having certain homology to the p55-IC and death domain thereof (Example 2). Accordingly, it is possible to construct the soluble, oligomeric TNF receptors of the invention by fusing the extracellular domain of the TNF receptor (as noted above) to the intracellular domain or the 'death domain' of the Fas/APOl receptor.
  • the oligomeric TNF receptors of the mvention are soluble by virtue of having only the soluble extracellular domain of the TNF receptor and the soluble intracellular domain or death domain thereof of either the p55-R TNF receptor or the Fas/APOl receptor, i.e. they do not contain the transmembranal (insoluble) domain of either type of receptor.
  • the construction of the above oligomeric TNF receptors of the invention are detailed herein below in Example 4.
  • the extracellular domain of the TNF receptor is capable of self-association, a situation that may not be desirable as it could interfere with the ability of the oligomeric receptor to bind to two or more TNF monomers of the TNF homotrimeric molecules or may lead to less than optimal binding of such TNF monomers. Accordingly, in such a situation, it is possible, by standard recombinant DNA procedures, to modify the extracellular domain of the TNF receptor by, for example, deleting or substituting one or more amino acid residues contained within the self-associating region to prevent such self-association.
  • Such modifications of the extracellular domain of the TNF receptor are thus also part of the present invention and are designated herein as analogs or derivatives of the extracellular domain of the TNF receptor.
  • the self- associating intracellular domain (IC) or death domain (DD) thereof of the p55-R receptor or the Fas/APOl receptor used in the oligomeric TNF receptors of the invention may also be analogs or derivatives thereof i.e. may be any modification of the p55-IC sequence or portions thereof including the death domain (p55DD). or any modification of the Fas/APOl intracellular domain (FAS-IC) sequence or portions thereof including the death domain (FAS DD), providing that these modifications yield a self-associating product.
  • the soluble oligomeric TNF receptors, analogs or derivatives thereof may be further modified by standard chemical means to provide salts and functional derivatives thereof for the purposes of preparing pharmaceutical compositions containing as active ingredients these TNF receptors of the invention.
  • the DNA sequences encoding the extracellular domain of the TNF receptor are obtained from existing clones of the entire TNF receptor, as is the intracellular domain or death domain thereof, and as is also the intracellular domain or death domain of the Fas/APOl receptor (see Example 2 and Example 5).
  • the DNA sequence of the desired extracellular domain is ligated to the DNA sequence of the desired intracellular domain or portion thereof including the death domain, and this fused product is inserted (and ligated) into a suitable expression vector under the control of the promoter and other expression control sequences.
  • the expression vector is introduced (transformation, transfection, etc.) into a suitable host cell, which then expresses the vector to yield the fusion product of the invention being the soluble self-associating TNF receptor molecules. These are then purified from the host cells by standard procedures to yield the final product being the soluble, oligomeric TNF receptors.
  • the preferred preparation of the fusion product encoding the extracellular domain and intracellular domain or portion thereof is by way of PCR technology using oHgonucleotides specific for the desired sequences to be copied from the clones encoding the entire TNF receptor molecule.
  • Other means are also possible, such as isolating the desired po ⁇ ions encoding the extracellular domain and the intracellular domain, by restriction endonucleases and then splicing these together in a known fashion, with or without modifications at the terminal ends of the restriction fragments to ensure correct fusion of the desired portions of the receptor (extracellular and intracellular domains or portions thereof).
  • the so-obtained fiision products are then inserted into the expression vector of choice.
  • the present invention also concerns soluble, oligomeric Fas/APOl (FAS) receptors containing the extracellular domain of the Fas/APOl receptor and the self- associating intracellular domain of the p55-R (p55-IC), the death domain thereof (p55DD), or the self-associating intracellular domain of the Fas/APOl receptor (FAS-IC) or the death domain thereof (FAS DD), or any analogs or derivatives thereof (see above).
  • FAS soluble, oligomeric Fas/APOl
  • Example 5 The constn ⁇ ction of these soluble, oligomeric FAS receptors is detailed in Example 5 herein below, using an available cloned full-length FAS receptor-encoding sequence as starting material and the appropriate oligonucleotides for PCR production of the desired extracellular and intracellular domains,- followed by ligation thereof to yield a fusion product, which is then inserted into a suitable expression vector.
  • prokaryotic or eukaryotic vectors and host cells may be used to produce the desired soluble, oligomeric FAS receptors, which can then be purified and formulated, as active ingredient, into a pharmaceutical composition.
  • the above soluble, oligomeric FAS receptors of the invention are intended for effective blocking of the Fas ligand, which may also exist as a trimer (similar to TNF, see above), each oligomeric receptor of the invention capable of binding two or possible more Fas ligands and thereby neutralize their activity.
  • the Fas ligand is known to be predominantly cell-surface associated but may also exist in a soluble form,
  • the oligomeric FAS receptors of the invention can bind to at least two monomers of this ligand and thereby neutralize more effectively (than monomeric FAS receptors) the activity of the Fas ligand.
  • the Fas ligand, and hence activation thereby of the FAS receptor has been implicated in a number of pathological states, particularly those relating to liver damage (apoptosis of hepatocytes, for example), including liver damage associated with hepatitis, as well as in autoimmune conditions, including lymphocyte damage (apoptosis) in HIV-infected humans (see. for example Ogasawara et al., 1993, Cheng et al., 1994).
  • the soluble, oligomeric FAS receptors of the invention are intended for blocking the activity of Fas ligand and may be used as active ingredient in pharmaceutical compositions for treating such Fas iigand-associated pathological states.
  • the present invention also concerns soluble, oligomeric receptors which have binding affinity for both TNF and FAS-R ligand, the so-called "mixed" TNF-R/FAS-R oligomeric receptors.
  • These mixed oligomeric receptors will contain at least one TNF-R extracellular domain and at least one FAS-R extracellular domain which are associated in the oligomeric receptor by virtue of each of these extracellular domains being fused to any one of the above-mentioned, self- associating, p55lC, p55DD, FAS IC or FAS DD
  • These mixed oligomeric receptors may be prepared by : (a) providing ary of the above noted fusion products which contain the extracellular domain of a TNF-R (p75 TNF-R, or preferably, p55 TNF-R) fused to any one of the self-associating intracellular domains p55 IC and FAS IC or any one of the self-associating 'dea
  • Another possibility for the preparation of the above mixed oligomeric receptors is by co-transforming suitable host cells with the above-mentioned expression vectors, one of which encodes the TNF-specific TNF-R fusion products and one of which encodes the FAS-R ligand- specific FAS-R fusion products. Following the expression of these different fusion products in the host cells, the mixed oligomeric (TNF-RTAS-R) receptors may be obtained by standard purification and selection procedures.
  • these mixed affinity oligomeric receptors are primarily for the neutralization of both TNF and FAS-R ligand when these are over-expressed endogenously or are at undesirably high levels following exogenous administration.
  • Recent evidence points to a likelihood that there exists a synergism in function between the FAS-R ligand (usually cell-surface associated) and TNF- ⁇ (which may also be cell-surface associated). Accordingly, in some instances it is desired to neutralize both of these ligands at the same point on the cell surface, i.e such a mixed-affinity receptor can block both the TNF binding to its receptor and the binding of FAS-R ligand to its receptor. Accordingly, these mixed-affinity receptors may be used as an active ingredient in pharmaceutical compositions for treating such conditions (see above) where both TNF and FAS- R ligand effects are undesirable.
  • any of the extracellular domains of the various receptors can be fused to the above-mentioned self-associating intracellular domains or portions thereof or to any other intracellular domains of the super family members also capable of self-association.
  • Expression of any of the recombinant proteins of the invention as mentioned herein can be effected in eukaryotic cells (e.g. yeast, insect or mammalian cells), using the appropriate expression vectors. Any method known in the art may be employed.
  • DNA molecules coding for the proteins obtained by any of the above methods are inserted into appropriately constructed expression vectors by techniques well known in the art (see Sambrook et al., 1989). Double-stranded cDNA is linked to ptasn.id vectors by homopoiymeric tailing or by restriction linking involving the use of synthetic DNA linkers or blunt-ended ligation techniques. DNA ligases are used to ligate the DNA molecules and undesirable joining is avoided by treatment with alkaline phosphatase.
  • an expression vector should comprise also specific nucieotide sequences containing transcriptional and translational regulatory information linked to the DNA coding for the desired protein in such a way as to permit gene expression and production of the protein.
  • RNA polymerase a promoter recognizable by RNA polymerase, to which the polymerase binds and thus initiates the transcription process.
  • promoters There are a variety of such promoters in use, which work with different efficiencies (strong and weak promoters). They are different for prokaryotic and eukaryotic cells.
  • the promoters that can be used in the present invention may be either constitutive, for example the int promoter of bacteriophage ⁇ , the bla promoter of the ⁇ -lactamase gene of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene of pPR325, etc., or inducible, such as the prokaryotic promoters including the major right and left promoters of bacteriophage ⁇ (? __ and PR), the trp, rec.A lac2. ig ., ompF. and ga
  • transcriptional and translational regulator ⁇ '' sequences may be employed, depending on the nature of the host They may be derived from viral sources, such as adenovirus, bovine papilloma virus. Simian virus, or the like, where the regulatory signals are associated with a particular gene which has a high level of expression. Examples are the TK promoter of Herpes virus, the SV40 early promoter, the yeast gal4 gene promoter, etc. Transcriptional initiation regulatory signals may be selected which allow for repression and activation, so that expression of the genes can be modulated
  • the DNA molecule comprising the nucieotide sequence coding for the fusion product proteins of the invention is inserted into a vector having the operably linked transcriptional and translational regulatory signals which is capable cf integrating the desired gene sequences into the host cell.
  • the cells which have been stably transformed by the introduced DNA can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may provide for phototrophy to an auxotropic host, biocide resistance, e.g. antibiotics, or heavy metals, such as copper, or the like.
  • the selectable marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co-transfection. Additional elements may also be needed for optimal synthesis of proteins of the invention These elements may include transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors incorporating such elements include those described by Okayama, H. (1 83).
  • the introduced DNA molecule will be incorporated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Factors of importance in selecting a particular plasmid or viral vector include : the ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E. coli, for example, pBR322, CoIEI, pSClOl, pACYC 184, etc. (see Maniatis et al.. 1982; Sambrook et al., 1989); Bacillus plasmids such as pC!94, pC22I, pT127, etc. (Gryczan, T.. (1982)); Streptomyces plasmids including pUlOl (Kendall, K.J et al., (1987)); Streptomyces bacteriophages such as 0C31 (Chater, K.F. et al..
  • Pseudomonas plasmids include BPV, vaccinia, SV40, 2-micron circle. etc., or their derivatives. Such plasmids are well known in the art (Botstein, D. et al., (1982); z>z
  • the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means : transformation, transfection, conjugation, protoplast fusion, eiectroporation, calcium phosphate-precipitation, direct microinjection. etc.
  • Host cells to be used in the invention may be either prokaryotic or eukaryotic.
  • Prefe ⁇ ed prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Se ⁇ atia, etc.
  • the most preferred prokaryotic host is E. coli.
  • Bacterial hosts of particular interest include E. coli K12 strain 294 (ATCC 31446), E. coli XI 776 (ATCC 31537), E. coli W31 10 (F", lambda * , prototropic (ATCC 27325)), and other enterobacterium such as Salmonella typhimurium or Serratia marcescens and various Pseudomonas species. Under such conditions, the protein will not be glycosylated.
  • the prokaryotic host must be compatible with the replicon and control sequences in the expression plasmid.
  • Preferred eukaryotic hosts are mammalian cells, e.g. human, monkey, mouse and Chinese hamster ovary (CHO) cells, because they provide post-translational modifications to protein molecules including correct folding or glycosylation at correct sites. Also yeasts cells can carry out post-translational peptide modifications including glycosylation.
  • Yeast recognizes leader sequences on cloned mammalian gene products and secretes peptides bearing leader sequences (i.e., pre-peptides).
  • the host cells After the introduction of the vector, the host cells are grown in a selective medium, which selects for the growth of vector-containing cells Expression of the cloned gene sequence(s) results in the production of the desired proteins.
  • Purification of the recombinant proteins is carried out by any one of the methods known for this purpose, i.e. any conventional procedure involving extraction, precipitation, chromatography, electrophoresis, or the like.
  • a further purification procedure that may be used in preference for purifying the protein of the invention is affinity chromatography using anti-TNF receptor monoclonal antibodies, which are produced and immobilized on a gel matrix contained within a column. Impure preparations containing the recombinant protein are passed through the column. The protein will be bound to the column by the specific antibody while the impurities will pass through. After washing, the protein is eluted from the gel by a change in pH or ionic strength.
  • salts of a carboxyl group include inorganic salts, for example, sodium, calcium, and salts with organic bases as those formed, for example, with amines, such as triethanolamine, arginine or *** »_ lysine.
  • Acid addition salts include, for example, salts with mineral acids and salts with organic acids.
  • “Functional derivatives” as used herein covers derivatives which may be prepared from the functional groups which occur as side chains on the residues or the N- or C- terminal groups, by means known in the a ⁇ , and are included in the invention as long as they remain pharmaceutically acceptable, i.e. they do not destroy the activity of the protein and do not confer toxic properties on compositions containing it. These derivatives include aliphatic esters or amides of the carboxyl groups, and N-acyl derivatives of free amino groups of O-acyl derivatives of free hydroxyl groups formed with acyl moieties (e.g. alkanoyl or carbocyclic aroyl groups). "Fractions” as used herein refers to any part or portion of the receptor, (intracellular or extracellular domains thereof), or of the proteins binding to the intracellular domain of the receptor, provided it retains its biological activity.
  • the present invention also relates to various pharmaceutical compositions comprising a pharmaceutically acceptable carrier and the various noted active ingredients of the invention or their salts, functional derivatives, or mixtures of any of the foregoing.
  • These compositions may be used in any of the conditions as noted herein, for example, in conditions where there is an over production of endogenous TNF, such as in cases of septic shock, cachexia, graft-versus host reactions, autoimmune diseases like rheumatoid arthritis, etc.
  • the way of administration can be via any of the accepted modes of administration for similar agents and will depend on the condition to be treated, e.g.
  • compositions of the invention when used to inhibit TNF effects they may be administered intravenously in case of septic shock or local injection in case of rheumatoid arthritis (for example, into the knee), or continuously by infusion, etc.
  • the compositions may also be used, for example, in cases of TNF intoxication caused by exogenous administration of excessive amount (overdoses) of TNF, e.g. in the case of cancer therapy or viral disease therapy.
  • the pharmaceutical compositions of the invention are prepared for administration by mixing the protein or its derivatives with physiologically acceptable carriers, stabilizers and excipients, and prepared in dosage form. e.g. by lyophilization in dosage vials.
  • the amount of active compound to be administered will depend on the route of administration, the disease to be treated and the condition of the patient. For example, local injection in case of inflammatory conditions of rheumatoid arthritis will require less active ingredient on a body weight basis than will intravenous infusion in case of septic shock.
  • this two-hybrid system is a yeast-based genetic assay to detect specific protein-protein interactions in vivo by restoration of a eukaryotic transcriptional activator such as GAL4 that has two separate domains, a DNA binding and an activation domain, which domains when expressed and bound together to form a restored GAL4 protein, is capable of binding to an upstream activating sequence which in turn activates a promoter that controls the expression of a reporter gene, such as lacZ or HIS3, the expression of which is readily observed in the cultured cells.
  • a eukaryotic transcriptional activator such as GAL4 that has two separate domains, a DNA binding and an activation domain, which domains when expressed and bound together to form a restored GAL4 protein, is capable of binding to an upstream activating sequence which in turn activates a promoter that controls the expression of a reporter gene, such as lacZ or HIS3, the expression of which is readily observed in the cultured cells.
  • the genes for the candidate interacting proteins are cloned into separate expression vectors.
  • the sequence of the one candidate protein is cloned in phase with the sequence of the GAL4 DNA-binding domain to generate a hybrid protein with the GAL4 DNA-binding domain
  • the sequence of the second candidate protein is cloned in phase with the sequence of the GAL4 activation domain to generate a hybrid protein with the GAL4-activation domain.
  • the two hybrid vectors are then co-transformed into a yeast host strain having a lacZ or HIS3 reporter gene under the control of upstream GAL4 binding sites.
  • the intracellular domains p55IC and p75IC were cloned, separately, into the vector pGBT9 (carrying the GAL4 DNA-binding sequence, provided by CLONTECH, USA, see below), to create fusion proteins with the GAL4 DNA-binding domain (similarly, the intracellular domain, FAS-IC and a portion of the 5 SIC, namely, the 55DD were also cloned into pGBT9 and used to isolate other IC-binding proteins, see Example 3 below).
  • clones encoding the full-length cDNA sequences of ⁇ 55 TNF-R (Schall et al., 1990) and ⁇ 75 TNF-R (Smith et al., 1990) were used from which the intracellular domains (IC) were excised as follows • p55IC was excised using the enzymes EcoRI and Sail, the EcoRI-Sall fragment containing the p55IC sequence was then isolated by standard procedures and inserted into the pGBT9 vector opened, in its multiple cloning site region (MCS), with EcoRI and Sail.
  • MCS multiple cloning site region
  • p75 IC was excised using the enzymes BspHI and Sail, the BspHI-Sall fragment containing the p75 IC sequence was then isolated by standard procedures and filled-in with the Klenow enzyme to generate a fragment which could be inserted into the pGBT9 vector opened with Smal and Sail.
  • the above hybrid (chimeric) vectors were then cotransfected (separately, one cotransfection with the ⁇ 55IC hybrid and one with the ⁇ 75 IC hybrid vector) together with a cDNA library from human HeLa cells cloned into the pGAD GH vector, bearing the GAL4 activating domain, into the HF7c yeast host strain (all the above-noted vectors, pGBT9 and pGAD GH carrying the HeLa cell cDNA library, and the yeast strain were purchased from Clontech Laboratories, Inc., USA, as a part of MATCHMAKER Two-Hybrid System, #PT1265- 1).
  • the co-transfected yeasts were selected for their ability to grow in medium lacking Histidine (His- medium), growing colonies being indicative of positive transformants.
  • the selected yeast clones were then tested for their ability to express the lacZ gene, i.e. for their LAC Z activity, and this by adding X-gal to the culture medium, which is catabolized to form a blue colored product by ⁇ -galactosidase, the enzyme encoded by the lacZ gene. Thus, blue colonies are indicative of an active lacZ gene.
  • the GAL4 transcription activator be present in an active form in the transformed clones, namely that the GAL4 DNA- binding domain encoded by one of the above hybrid vectors be combined properly with the GAL4 activation domain encoded by the other hybrid vector.
  • Such a combination is only possible if the two proteins fused to each of the GAL4 domains are capable of stably interacting (binding) to each other.
  • the His 4" and blue (LAC ZY) colonies that were isolated are colonies which have been cotransfected with a vector encoding p55IC and a vector encoding a protein product of human HeLa cell origin that is capable of binding stably to p55 IC, or which have been transfected with a vector encoding p75IC and a vector encoding a protein product of human HeLa cell origin that is capable of binding stably to p75 IC
  • the plasmid DNA from the above His ⁇ , LAC 7 ⁇ yeast colonies was isolated and eiectroporated into E. coli strainHBlOl by standard procedures followed by selection of Leu + and Ampicillin resistant transformants, these transformants being the ones carrying the hybrid pGAD GH vector which has both the Amp ⁇ - and Leu ⁇ coding sequences Such transformants therefore are clones carrying the sequences encoding newly identified proteins capable of binding to the p551C or p75IC. Plasmid DNA was then isolated from these transformed E. coli and retested by :
  • hybrid plasmids (hybrid pGTB9 carrying either the p55lC or p75IC sequences) into yeast strain HF7 as set forth hereinabove.
  • vectors carrying irrelevant protein encoding sequences e.g. pACT- larain or pGBT9 alone were used for cotransformation with the p55IC -binding protein or p75IC- binding protein encoding plasmids.
  • the cotransformed yeasts were then tested for growth on His" medium alone, or with different levels of 3-aminotriazole; and
  • LAC Z activity in liquid culture was assessed after transfection of the GAL4 DNA-binding and activation-domain hybrids into the SFY526 yeast hosts which have a better LAC Z inducibility with the GAL4 transcription activator than that of the HF-7 yeast host cells.
  • the plasmids and hybrid encoding the GAL4 DNA-binding domain and GAL4 activation domain are as follows ;
  • DNA-binding domain hybrids pGBT9-IC55 : full-length intracellular domain of the p55-TNF-R (p55IC)
  • pACT-Lamin irrelevant protein - lamin.
  • pGBT9 vector alone
  • pGBT9-IC75 full-length intracellular domain of the p75-TNF-R (p75IC)
  • 55.1 and 55.3 correspond to fragments of the intracellular domain of the p55-TNF-R.
  • 55.11 is the novel protein associating with the p55-TNF-R 75.3 and 75.16 are the novel proteins associating with the p75-TNF-R.
  • FIG. 1(c) depicts the partial sequence of the cDNA encoding protein 75.16.
  • Fig. 1(d) there is shown the deduced amino acid sequence of the protein 55.11, as deduced from the nucieotide sequence of Fig. 1(a).
  • 55.11 cDNA [nucleotides 1-924 (Fig. 1(a)) which code for amino acids 1-308 (Fig. 1(d))] was obtained by standard procedures, namely, by cloning by PCR from a human fetal liver cDNA library (for more details, see below). The full nucieotide sequence of 55.11 (Fig. 1(a)) was determined in both directions by the dideoxy chain termination method.
  • Glutathione S-transferase (GST) fusions with p55-IC (GST-p55lC) and with p55-lC truncated below amino acid 345 (GST-p55IC345) were produced and adsorbed to glutathione-agarose beads as described in Example 2 below (see also Smith and Corcoran, 1994; Frangioni and Neel, 1993).
  • the cDNAs of 55.11 (1-2863 nucleotides, i.e. the full-length 55.11 cDNA), of FLAG-55.11, and of luciferase were expressed in HeLa cells.
  • FLAG-55.11 is the region extending between residues 309 and 900 in the 55.11 protein (the partial cDNA of 55.1 1 (nucleotides 925-2863), originally cloned by the two hybrid screen), N-linked to the FLAG octapeptide (Eastman Kodak, New Haven. Ct., U.S.A.). Expression of the fusion proteins was accomplished using a tetracycline-controlled expression vector (HtTA-1) in a HeLa cell clone that expresses a tetracycline-controlled transactivator (see Example 2 below, and Gossen and Bujard, 1992).
  • HtTA-1 tetracycline-controlled expression vector
  • the immunoprecipitations of 55.11 and FLAG-55.1 1 were achieved using a rabbit antiserum (diluted 1 :500) raised against a GST fusion protein containing the region of 55.11 that extends between amino acids 309 and 900 and a mouse monoclonal antibody against the FLAG octapeptide (M2; Eastman Kodak; 5 ⁇ g ml of cell lysate).
  • M2 Westman Kodak
  • various deletion mutants of 55.11 were also constructed and fused to the "activation domain" in the GAL4AD vector (e.g. mutants of 55.11 having only residues 309-680 and 457-900).
  • the binding of the various 'binding domain' constructs to the various 'activation domain 1 constructs was examined in transfected SFY526 yeast cells. The binding was assessed by a two-hybrid ⁇ -galactosidase expression filter assay.
  • the non-relevant proteins SNF1 and SNF4 served as positive controls for the 'binding domain' and 'activation domain' constructs, respectively; the empty Gal4 (pGAD-GH) and VP16 (pVP16) vectors served as negative controls for the 'activation domain' constructs; 2nd the empty Gal4 (pGBT9) vector served as a negative control for the Tending domain' constructs.
  • the results of the assay are set forth in Table 2 below in which the symbols "-r ⁇ -" and " ⁇ +” indicate the development of strong color within 20-60 min of initiation of the assay, respectively (positive binding results); and "-" indicates no development of color within 24h of commencement of the assay (negative results). Blank spaces in the Table indicate binding assays not tested.
  • the 55.11 protein bound to a truncated p55-IC from which the death domain had been deleted (construct 206-328 in Table 2), more effectively than to nontruncated p55-IC. It also bound to an even further C terminally truncated construct (construct 206-308) and to a construct from which both the death domain and a membrane proximal part were deleted (construct 243- 328). However, the 55.11 protein did not bind to a construct that was N-terminally truncated down to amino acid 266 (Table 2). These findings indicate that the binding site for 55,11 is located in the region that extends between residues 243 and 308 of p55-IC and that the N terminus of this binding site is between residues 243 and 266.
  • the cell lines examined were HeLa, CEM, Jurkat, and HepG2 cells derived from human epithelial carcinoma, an acute lymphoblastic T cell leukemia, an acute T cell leukemia, and a hepatocellular carcinoma, respectively.
  • the 3 kB cDNA hat encompassed both these portions was effectively expressed m transfected HeLa cells (see below) yielding a protein of about 84 kDa, which suggests that the 3 kB cDNA contains a translational start site.
  • Lysates of the transfected cells were immunoprecipitated with antibodies against the 55.1 1 protein when the full-length 55.11 protein was used for binding the GST-fusion proteins, or with antibodies against the FLAG octapeptide when the FLAG-55.11 fusion product was used for binding the GST-fusion proteins.
  • the proteins were analyzed by SDS- polyacrylamide gel electrophoresis (SDS-PAGE; 10% acrylamide), followed by autoradiography.
  • SDS-PAGE SDS- polyacrylamide gel electrophoresis
  • Figs. 3 A and B are shown reproductions of the autoradiograms of the above SDS-PAGE gels, in which Fig.
  • FIG. 3A depicts the binding of the full-length 55.11 protein (55.11 -full) to the various GST-fusion proteins; and in which Fig 3B depicts the binding of the Flag-55.11 fusion product to the various GST-fusion proteins.
  • Fig. 3 A there is shown in the extreme right hand lane a control immunoprecipitate of lysates of cells transfected with only the full-length 55.11 and immunoprecipitated with the a_nti-55.l l antibodies ( ⁇ .55.11 Abs).
  • Fig. 3B there is shown in the extreme right band lane a control immunoprecipitate of lysates of cells transfected with only the FLAG-55.1 1 and immunoprecipitated with the anti-FLAG antibodies ( ⁇ FLAG Abs).
  • the protein encoded by the full-length 55.11 cDNA can be expressed in HeLa cells and it binds to fusion proteins that contained the full p55-IC (GST-p55IC) or a truncated p55-lC that Jacked most of the death domain (GST- p55IC345) (Fig. 3A).
  • the full-length 55.11 protein did not bind to GST alone (control).
  • the full-length 55.11 cDNA has been cloned and sequenced (see nucieotide sequence in Fig. 1(a)) and the full amino acid sequence of 55.11 has been deduced from the cDNA sequence (see amino acid sequence in Fig. 1(d)).
  • Data bank GenBankTM/EMBL DataBank
  • searches revealed that parts of the sequence of the human 55.11 cDKA (accession numbers T03659. Z19559, and F09128) and its mouse homologue (accession numbers X80422 and Z31147) have already been determined during arbitrary sequencing of cDNA libraries.
  • a cDNA sequence (accession number U18247) that encodes for a human protein of 596 amino acids present in cultures of human hepatoma HC10 cells is similar to that of 55.11.
  • This hepatoma protein lacks an N terminal portion (amino acids 1-297) corresponding to that of 55.11 and also differs from 55.11 at the regions that correspond to residues 297-377 and residues 648-668 in 55.11.
  • the searches of the data bank also revealed that proteins with very high sequence homology to 55.11 exist in Saccharomyces cerevisiae (yeasts), Arabidopsis thaliana (plants) and Caenorhabditis elegans (worms).
  • 55.11 appears to fulfill an evolutionary conserved function.
  • YHR027c In the yeasts, there are two known proteins (the open reading frame YHR027c and SEN3) whose DNA sequences resemble that of 55.11. The sizes of both are close to that of 55.11. YHR027c is known only by the sequencing of a genomic clone while SEN3 has been cloned as a cDNA, The sites within 55.11 that are similar to those in SEN3 correlate to the sites of its similarity to YHR027c, although much more similarity is evident between 55.1 i and YHR027c than between 55.11 and SENS.
  • Fig. 4 there is shown schematically a comparison of the deduced amino acid sequence of human 55.11 to that of the above-mentioned, related proteins present in lower organisms.
  • the sequences that are compared are the sequences of amino acids predicted for : the 55.11 cDNA (see Fig.
  • an open reading frame within a cosmid derived from the 8th chromosome of Saccharomyces cerevisiae (nucleotides 21253-24234, accession number U10399); SEN3, the cDNA of a Saccharomyces cerevisiae protein (accession number L06321); a partial cDNA of a protein of the plant Arabidopsis thaliana (accession number T21500), and a partial cDNA of a protein of the nematode Caenorhabditis elega (accession number D27396).
  • the 'KEKE' sequence in 55.11 is marked with a solid line and the sequence AYAGS(x)gLL with broken lines.
  • the sequences were aligned using the PILETJP and PRETTYBOX programs of the GCG package. Gaps introduced to maximize alignments are denoted bv dashes.
  • conserved amino acid sequence motifs were not discerned within the protein encoded for by 55.11, except for a repetitive 'KEKE' sequence that extends between Lys 614 and Glu 632 (underlined in Fig. 4).
  • Such 'KEKE' sequences which are present in many proteins, including proteasonal subunits and chaperonins, may promote association of protein complexes (Reaiini et al., 1994).
  • a sequence AYAGS(x)gLL appears twice in the 55.11 protein (at sites 479 590. see Fig. 4); no functional significance for this sequence has yet been described
  • this region does not contain the RPMI and RPM2 proline-rich motifs present in several other cytokine receptors (O'Neal and Yu- Lee, 1993)
  • the region that extends between residues 243 and 266, whose deletion abolishes the binding of p55-R to 55 1 1 see (b) and (d) above and Table 2)
  • two of the serines and two of the threonines are followed by proline residues, which makes them potential sites for phosphorylation by MAP kinase, CDC2, and other proline-dependent kinases (Seger and Krebs, 1995). Phosphorylation of this site in the receptors might affect its binding to the 55.11 protein.
  • association (binding) of 55.1 1 with the intracellular domain of p55-TNF-R affects or is involved in : (i) the signaling for these above noted or other TNF effects, (ii) the folding or processing of the protein (as suggested by the similarity of 55.11 to a subunit of the 26S proteasome), or (iii) the regulation of the activity or expression of p55-TNF-R
  • p55-IC is capable of self-association.
  • Two of these clones were identical, containing an insert which encodes for amino acids 328-426 (designated as clone 55.1 encoding protein fragment 55.1 of the p55IC).
  • the third contained a longer insert, encoding for amino acids 277-426 (designated as clone 55.3 encoding protein fragment 55.3 of the p55IC).
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • chimeras were constructed, cloned and expressed by standard methods. Following their expression, the assessment of the self-interaction of the p55-R intracellular domain (p55IC) by determining the interaction of the above bacterially-produced chimeric proteins GST-IC55 (Mr - 5lkD) and MBP-IC55 (Mr - 67 kD) with each other. Equal amounts of the GST-IC55 chimera (samples of lanes 1-4 in Fig.
  • buffer I (20mM Tris-HCl, pH 7.5, lOOmM KC1, 2mM CaCl 2 , 2mM MgCl 2 , 5mM
  • the p55-IC, the Fas receptor and CD 40 can all be stimulated by antibodies against their extracellular domain. Their stimulation could be shown to co elate with the ability of the antibodies to cross-link the receptors. It therefore seems that the signaling is initiated as a consequence of interaction of two or more intracellular domains imposed by aggregation of the extracellular domains. Involvement of such interaction in the initiation of signaling of these receptors was also indicated by studies (Brakebusch et al., 1992) showing that expression of receptors made nonfunctional by mutation of their intracellular domain, had a "dominant negative" effect on the function of co-expressed normal receptors.
  • the propensity of the p55-IC to self associate indicates that this domain plays an active role in its induced aggregation. Moreover, this activity of the p55-IC seems to suffice for initiating its signaling, since when expressed independently of the rest of the receptor molecule, it can trigger cell death in the absence of TNF or any other exterior stimuli. Nevertheless, when expressed as the full length receptor, the p55-TNF-R does not signal, unless stimulated by TNF.
  • TNF actually overcomes some inhibitory mechanisms, which prevent spontaneous association of the intracellular domains, and this inhibition is due to the linkage of the p55-lC to the rest of the receptor molecule.
  • the inhibition may be due to the orientation imposed on the intracellular domain by the transmembrane and extracellular domain, to association of some other proteins with the receptor or perhaps just due to restriction of the amounts of receptors that are allowed to be placed in the plasma membrane, Of note, this control mechanism should be rather effective, since according to some estimations, the binding of even just one TNF molecule to a cell suffices for the triggering of its death.
  • Spontaneous signaling independent of ligand can result in extensive derangement of the process controlled by this receptor.
  • the best known example is the deregulation of growth factor receptors. Mutations due to which they start signaling spontaneously, for example those that cause them to aggregate spontaneously, play an important role in the deregulated growth of tumor cells. TNF effects, when induced in excess, are well known to contribute to the pathology of many diseases. The ability of free intracellular domains (p55ICs) of the p55-TNF-R to signal independently of TNF may contribute to such excessive function.
  • Glutathione S-transferase GST
  • GST-p55-IC glutathione S-transferase-p55-IC fusion protein
  • GST glutathione S-transferase-p55-IC fusion protein
  • Incubation with MBP fusion proteins was carried out in a buffer solution containing 20 mM Tris-HCl, pH 7.5, 100 mM KG, 2 mM CaCb, 2 mM MgCl2, 5 mM dithiotreitol, 0.2% Triton XI 00, 0.5 mM phenyl-methyl-sulphonyl-fluoride and 5% (v/v) glycerol or, when indicated, in that same buffer containing 0.4 M KG, or 5 mM EDTA instead of MgC .
  • MBP fusion proteins Association of the MBP fusion proteins was assessed by SDS polyacrylamide gel electrophoresis (10% acrylamide) of the proteins associated -with the glutathione-agarose beads, followed by Western blotting. The blots were probed with rabbit antiserum against MBP (produced in our laboratory) and with horseradish-peroxidase-Iinked goat-anti-rabbit immunoglobulin.
  • HeLa cells expressing the tetracycline-controlled transactivator developed by Gossen and Bujard were grown in Dulbecco's modified Eagle's medium, containing 10% fetal calf serum, 100 u/ml penicillin, 100 ⁇ g/ml streptomycin and 0.5 mg/ml neomycin.
  • cDNA inserts encoding the p55-R or parts thereof were introduced into a tetracycline-controlled expression vector (pUHD 10-3, kindly provided by H, Bujard).
  • the cells were transfected with the expression construct (5 ⁇ g DNA/6 cm plate) by the calcium phosphate precipitation method (Ausubel et al., 1994). Effects of transient expression of the transfected proteins were assessed at the indicated times after transfection in the presence or absence of tetracycline (1 ⁇ g/ l).
  • Clones of cells stably transfected with the human p55-IC cDNA in the p ⁇ HD 10-3 vector were established by transfecting the cDNA to HtTA-1 cells in the presence of tetracycline together with a plasmid conferring resistance to hygromycin, followed by selected for clones resistant hygromycin (200 ⁇ g/ml). Expression of the cDNA was obtained by removal of tetracycline which was otherwise maintained constantly in the cell growth medium.
  • TNF receptor expression in samples of 1x10 ⁇ cells was assessed by measuring the binding of TNF, labeled with ⁇ 5j b t h e chloramine-T method, as previously described (Holtmann and Wallach, 1987). It was also assessed by ELISA, performed as described for the quantification of the soluble TNF receptors (Aderka et al., 1991), except for the use of RIP A buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS and 1 M EDTA) to lyse the cells (70 ⁇ l/10 6 cells) and to dilute the tested samples. The soluble form of the p55-R, purified from urine, served as the standard. (b) Mutational analysis of the intracellular domain of the p55-R (p55-IQ to determine the regions of the D55-IC involved in its self-association
  • p55-IC can self-associate and trigger cytotoxic effects on cells, and there are portions of the p55-IC, which themselves were capable of binding to the full-length p55-IC
  • one of the portions of the p55-IC (designated as protein fragment 55.1 in Example 1 above) was identified that was capable of binding strongly to the full length p55-IC. This portion was sequenced and was observed to contain the amino acid residues 328-426 of the p55-TNF-R, which are within the p55-!C It has further been discovered (see below) that the above portion, protein fragment 55.1, is itself capable of self-association and of triggering cytotoxic effects on cells.
  • this portion of the p55-IC has been called the 'death domain', and is located in the region between amino acid residues 328-426 of the human p55-R most likely consisting of amino acid residues between about residue 328 and 414 thereof.
  • Fas-IC which signals for cell death by a sequence motif related to the p55-R 'death domain', self- associates, and associates to some extent with the p55-IC.
  • CD40-IC that provides growth stimulatory signals (even though also containing a sequence resembling the 'death domain')
  • p75-lC that bears no structural resemblance to p55-IC, do not self associate, nor do they bind p55-IC or Fas-IC.
  • Table 3 above shows the quantitative assessment of the interaction of Gal4 hybrid constructs encompassing the following proteins : the intracellular domain of human p55-R and its various deletion mutants (residues numbered as in (Loetscher et al , 1990)); the intracellular domains of mouse p55-R (residues 334-454, numbered as in (Goodwin et al., 1991)); mouse Fas/APOl (Fas-IC, 166-306, numbered as in (Watanabe-Fukanaga et al., 1992)); human CD40 (CD40-IC, 216-277, numbered as in (Stamenkovic et al., 1989)); and human p75 TNF receptor (p75-IC, 287-461, numbered as in (Smith et al., 1990)).
  • SNF1 and SNF4 were used as positive controls for association (Fields and Song, 1989), and lamin as a negative control (Bartel et al., 1993).
  • Proteins encoded by the Gal4 DBD constructs (pGPT9) are listed vertically; those encoded by the Gal4 AD constructs (pGAD-GH), horizontally.
  • the two deletion mutants denoted by asterisks were cloned in a two-hybrid screen of a HeLa cell cDNA library (Clontech, Palo Alto, Ca., U.S.A.) using p55-IC cloned in pGBT9 as "bait". In that screen, four of about 4x10 6 cDNA clones examined were positive.
  • ⁇ -galactosidase expression data are averages of assays of two independent transformants and are presented as amount of ⁇ - galactosidase product; (a unit of activity being defined as OD420 tiroes 10 ⁇ divided by ODg ⁇ o of the yeast culture and reaction time, in minutes).
  • the detection limit of the assay was 0.05 units. Variation between duplicate samples were in all cases less that 25% of the average (not tested).
  • GST p55-IC-glutathione-S-transferase
  • MBP p55-lC-maltose binding protein
  • Fig. 7 depicts the ligand- independent triggering of a cytocidal effect in HeLa cells transfected with p55-R, its intracellular domain (p55-IC) or parts thereof (including the 'death domain*).
  • Fig. 7 there is shown schematically, the various DNA molecules encoding the different types of TNF receptors included in the vectors with which the HeLa cells were transfected (extreme left hand side of Fig. 7); and the expression (left and middle bar graphs) and the viability (right bar graph) in HeLa cells expressing transiently the various full-length p55-R (p55-R), p55- IC or parts of p55-IC or, as a control, Iuciferase (LUC) (each being depicted at the extreme left side of Fig. 7), using a tetracycline-controlled expression vector.
  • p55-R full-length p55-R
  • p55-IC p55-IC
  • LOC Iuciferase
  • the open bar graphs represent cells transfected in the presence of tetracycline (1 ⁇ g/ml), which inhibits expression; and the filled bar graphs (left, middle and right) represent cells transfected in the absence of tetracycline.
  • TNF receptor expression was assessed 20h after transfection, both by ELISA, using antibodies against the receptor's extracellular domain (see schematic illustration on the left side of Fig. 7), and by determining the binding of radiolabeled TNF to the cells (middle).
  • the cytocidal effect of the transfected proteins was assessed 4Sh after transfection. Data shown are from one of three experiments with qualitatively similar results, in which each construct was tested in duplicate. ND - not determined.
  • panel B of Fig. 8 there is shown a reproduction of a Northern blot representing the methylene blue staining of 18S rRNA in each of the samples shown in panel A of Fig. 8.
  • the 'death domain 1 thereof is primarily responsible for signaling the induction of the intracellular processes leading to the triggering of cytotoxicity within the cells, whilst the other effects, e.g the signaling leading to the induction of IL-8 gene expression, are likely due to other regions of the p55-IC as well, following the self-association thereof. It is therefore possible that different regions of the p55-IC are responsible for the different TNF-induced effects (e.g. cytotoxicity, IL-8 induction) within cells, these effects being a consequence of the intracellular signaling upon self-association of the p55- IC
  • the p55-IC can induce in a ligand (TNF)-independent fashion, the triggering of other intracellular effects e.g. IL-8 induction, means that the p55-lC or specific portions thereof may be used as a highly specific tool for bringing about such effects in cells or tissues that it is desired to treat, without the need for treating such cells or tissues with TNF.
  • TNF ligand
  • treatment with TNF especially at high dosages can lead to undesirable side-effects due to the number of intracellular effects induced systemically by TNF following its binding to its receptors.
  • the p55-IC can mimic specific other TNF-induced effects (besides cytotoxicity), e.g.
  • IL-8 induction opens the way for introducing in a cell- or tissue-specific manner, p55-IC or specific portions thereof, ⁇ vhich will be capable of signaling for the induction of specific desired intracellular effects, e.g. IL-8 induction, and thereby overcome the systemic side-effects often observed during TNF treatment.
  • cytotoxic activity of the intracellular domains of the p55 TNF-R and FAS- R (p55IC and FAS-IC) it has now also been further elucidated that both the p55IC. its 'death domain' (p55DD) and the FAS-IC are capable of a ligand-independent triggering of a cytocidal effect in HeLa cells.
  • p55DD 'death domain'
  • FAS-IC are capable of a ligand-independent triggering of a cytocidal effect in HeLa cells.
  • HeLa cells were transfected with expression vectors containing various constructs of either the full-length p55-TNF-R, portions thereof including the p55IC and p55DD or the FAS-IC.
  • Fig. 9 A and B
  • the constructs used for transfecting the HeLa cells are shown schematically in the left hand panels; the results of the TNF or FAS receptor expression are shown graphically in the two middle panels (second and third panels from the left), and the results of transfected cell viability are shown graphically in the right hand panels.
  • Fig. 9A and B the constructs used for transfecting the HeLa cells are shown schematically in the left hand panels; the results of the TNF or FAS receptor expression are shown graphically in the two middle panels (second and third panels from the left), and the results of transfected cell viability are shown graphically in the right hand panels.
  • FIG. 9A there is shown the results of transfected HeLa cells transiently expressing the full-length p55-R, p55-lC or parts thereof, or as a control, luciferase (LUC), in all cases using a tetracycline-controlled expression vector.
  • Fig. 9B there is shown the results of transfected HeLa cells transiently expressing FAS-IC alone or together with the p55-R, using a tetracycline-controlled expression vector.
  • the open bars represent cells transfected in the presence of tetracycline (1 ⁇ g/ml), which inhibits expression
  • the closed bars represent cells transfected in the absence of tetracycline.
  • TNF receptor expression was assessed 20h after transfection, both by ELISA using antibodies against the extracellular domain of the receptor (see left hand panels), and by determining the binding of radiolabeled TNF to the cells (middle panels).
  • the cytocidal effect of the transfected proteins was assessed 48h after transfection.
  • the data shown are from one of three experiments with qualitatively similar results in which each construct was tested in duplicate
  • the designation *ND' in Figs. 9A and B means not determined. From the results shown in Figs. 9A and B it is apparent that expression of only the p55IC results in even greater cytotoxicity. Significant cytotoxicity also occurs when expressing just the death domain (p55DD).
  • Figs. 10-12 there is shown schematically the partial and preliminary nucieotide sequence of cDNA clones, called F2, F9 and DD11, respectively.
  • Clones F2 and F9 were isolated by screening a murine (mouse) embryonic library using the murine FAS-IC as "bait".
  • Fig. 10 there is shown schematically the partial nucieotide sequence from the F2 cDNA that has been sequenced.
  • Fig. 11 there is shown schematically the partial nucieotide sequence of 1724 bases from the F9 cDNA that has been sequenced.
  • F2 and F9 Analysis of the binding capability of the protein encoded by clones F2 and F9 (F2 and F9, respectively) has shown tha : (a) F2 interacts strongly with human p55IC and p55DD and with murine FAS-IC, while it interacts weakly with non-relevant (control) proteins SNF1 and La in as well as relevant protein, human FAS-IC.
  • F9 interacts strongly with human p55-IC and murine FAS-IC, while it interacts weakly with human FAS-IC (relevant protein) and irrelevant proteins SNF1 and Lamin.
  • F2 nor F9 interacted at all with human p75IC, pGBT9 (empty bait vector), or human CD-40.
  • F2 and F9 represent new proteins having binding specificity for both FAS-IC and p55IC.
  • Clone DD11 was isolated by screening a human HeLa library using the human p55DD as "bait".
  • Fig. 12 there is shown schematically the partial nucieotide sequence of 425 bases from the D 11 cDNA that has been sequenced.
  • the DD11 clone has an approx. length of 800 nucleotides.
  • the full length of the transcript capability of the protein encoded by clone DD11 has shown that DD11 interacts strongly with the p55DD (a.a. 326-414) (see Fig. 9) and does not interact with deletion mutants of this domain, e.g. a.a. 326-404.
  • DDll also interacts with mouse and human FAS-IC and to some extent also with Lamin.
  • DD1 1 does not interact at all with SNF1 nor with pGBT9 (empty bait vector).
  • DD11 is also not found in the 'Gene Bank' and Protein Bank' databases.
  • DD11 represents a p55 IC (p55DD) and FAS-IC specific binding protein.
  • TNF receptors will be fusion ' proteins having essentially all of the extracellular domain of the p55-R fused to essentially all of the intracellular domains or 'death domains' thereof of the p55-R or Fas/APOl .
  • fusion constructs will be devoid of the transmembranal domain of the p55-R (or FAS/APO1) and hence will be soluble.
  • these fusion constructs will be capable of ohgomerization to provide at least dimers (and possibly also higher order multimers) of the p55- R.
  • such dimeric TNF receptors (p55-R) will be capable of binding to at least two TNF monomers of the naturally-occurring TNF homotrimer to provide a soluble TNF receptor which binds more avidly to its ligand (homotrimeric TNF).
  • p55 TNF receptor fusion proteins will be constructed each of which will be capable of oligomerization and will be soluble :
  • the TNF monomer binding capability is provided by the EC55 portion while the oligomerization (or at least dimerization) of each kind of fusion protein is provided by its 'tail' region being any of the p55IC, DD55, ICFAS or DDFAS portions.
  • any suitable bacterial; bacteriophage, or animal virus expression vector (cloning vehicle or plasmid designed for expression of the inserted DNA of choice) may be employed into which will be inserted in one or more stages the DNA encoding the EC55 and one of the 'tails' being the p55-lC. DD55. ICFAS or DDFAS.
  • the so-inserted DNA encoding each of the fusion proteins will be placed under the control of the various expression control sequences of the cloning vehicle or plasmid such as promoters, ribozyme binding sites, transcriptional factor binding sites, etc.
  • expression control sequences will be chosen depending on the type of expression vector chosen and hence the type of host cell (eukaryotic or prokaryotic) in which it is desired to express the fusion proteins of the invention.
  • Preferred host cells are eukaryotic, in particular, mammalian.
  • oligonucleotides for use in PCR will be constructed by standard means, the oligonucleotides being :
  • Plasmids containing the cloned full-length p55-R and Fas/APOl receptors which we have in our laboratory (see also co-pending EP568925 and Examples 1-3 above) will be subjected to the following manipulations to yield the DNA fragments encoding each of the fusion proteins, which DNA fragments are then ligated into the above noted expression vector of choice : CO To produce the DNA fragment coding for EC55 which is a component of all 4 fusion proteins. PCR is performed on a plasmid bearing cDNA of human ⁇ 55 using the above oligonucleotide nos. 1 and 2 (size of fragment 640 bp).
  • PCR is performed on a plasmid bearing cDNA for human p55 using oligonucleotide nos. 3 and 4, to obtain a DNA fragment coding for 1C55 (size 677 bp) which is then mixed with EC55 digested by Sal I and ligated by blunt end ligation into any expression vector for mammalian cells under the control of an appropriate promoter.
  • the orientation of the inse ⁇ ed EC55--IC55 in the vector is verified by restriction digestion and by sequencing.
  • IC FAS is produced by PCR on a plasmid with cDNA for FAS using oligonucleotide nos. 5 and 6, to obtain a fragment (size 448 bp) which is then cut by Sal I and mixed with EC55 cut by Sail, and subsequently is blunt ligated into a mammalian expression vector under the control of an appropriate promoter.
  • the orientation of the inserted EC55-IC FAS in the vector is verified by restriction digestion and by sequencing.
  • a DNA fragment is produced with the
  • DD55 sequence by PCR in cDNA for human p55 using oligonucleotide nos. 7 and 4.
  • the product with a size of 314 bp is cut by Sail and mixed with EC55 cut by Sail, and subsequently blunt ligated into the mammalian expression vector. Orientation of the inserted EC55--DD55 in the
  • 5 vector is verified by restriction digestion and by sequencing.
  • a DNA fragment with DD FAS is produced by PCR on cDNA for FAS using oligonucleotide nos, 6 and 8.
  • the product with a size of 332 bp is cut with Sail, and mixed with EC55 cut by Sal I and subsequently blunt ligated into the mammalian expression vector. Orientation of the EC55--DD FAS is then verified by
  • the above expression vectors will then be introduced by standard methods into suitable mammalian cells (e.g. Chinese Hamster Ovary (CHO) or Monkey Kidney (COS) cells) for the purposes of expression.
  • suitable mammalian cells e.g. Chinese Hamster Ovary (CHO) or Monkey Kidney (COS) cells
  • the so-expressed fusion proteins will then be purified by standard methods (see co-pending EP308378; EP398327; and EP568925).
  • the 15 purified fusion proteins will then be analyzed for their ability to oligomerize (and the extent thereof, i e. whether they form dimers or higher order multimers) and for their ability to bind TNF (and the affinity or avidity of binding thereof).
  • the FAS ligand binding capability is provided by the EC FAS portion, while the oligomerization (or at least dimerization) of each kind of fusion protein is provided by its 'tail' region being any of the p55-IC.
  • DNA fragments encoding the above fusion proteins and expression vectors containing them will be as detailed in Example 4, except different appropriate oligonucleotides (not shown) will be used for the preparation of the EC FAS fragment to be ligated to any of the above noted 'tail' regions. Subsequently, the expression vectors will be
  • the resulting expressed fusion proteins will be purified and tested for their ability to oligomerize (and the extent thereof, i.e. whether they form dimers or higher order multimers) and for their ability to bind the FAS ligand (and the affinity or avidity of binding thereof).
  • fusion products may be utilized in the following procedure ; i) Providing a fusion product as set forth in Example 4, which contains the extracellular domain of a TNF-R (p75 TNF-R or p55 TNF-R) fused to any one of : the ⁇ 55 IC, FAS-IC, p55 D or FAS DD; ii) Providing a fusion product as set forth in Example 5, which contains the extracellular domain of Fas-R fused to any one of : p55 IC, FAS-IC, p55 DD or FAS-DD; and iii) mixing any one of the fusion products of i) with any one of the fusion products of ii) to provide a new dimeric (or higher order oligomeric) receptor which has both the extracellular domains of
  • the fusion products of i) and ii) may be provided separately, namely, from their purification from transformed cells in which they were produced, and then mixed in vitro to obtain the mbced affinity receptors.
  • the host cells may be co ⁇ transfected with vectors carrying sequences encodmg both types of fusion products, in which case, the mixed affinity receptors may be obtained directly from the co-transfected cells.
  • the actual oligomerization of the fusion products into oligomeric receptors may take place within the cells or during or following the purification procedure to obtain the fusion products expressed in the cells.
  • any standard method may be utilized, for example, affinity chromatography procedures in which antibodies against the TNF-R and FAS-R extracellular domains are used in sequential chromatographic steps to select for those receptors having both types of extracellular domain.

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Abstract

Nouvelles protéines qui se lient aux domaines intracellulaires des TNF-Rs (récepteurs de la famille du facteur de nécrose tumorale) p55 et p75 et de Fas-R, capables de moduler la fonction des TNF-Rs p55 et p75 et de Fas-R, et séquences d'ADN les codant. La présente invention concerne également de nouveaux TNF-Rs oligomères solubles, des Fas-R oligomères et des récepteurs oligomères présentant un mélange de TNF-Rs et de Fas-Rs. La présente invention concerne en outre des procédés de préparation et d'utilisation de toutes les substances susmentionnées.
EP95919787A 1994-05-11 1995-05-11 Modulateur de recepteurs de la super-famille tnf/ngf et recepteurs oligomeres solubles de la super-famille tnf/ngf Withdrawn EP0759984A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
IL10963294 1994-05-11
IL109632A IL109632A (en) 1994-05-11 1994-05-11 Modulators of the function of tnf receptors
IL11112594A IL111125A0 (en) 1994-05-11 1994-10-02 Soluble oligomeric tnf/ngf super family ligand receptors and their use
IL11112594 1994-10-02
PCT/US1995/005854 WO1995031544A1 (fr) 1994-05-11 1995-05-11 Modulateur de recepteurs de la super-famille tnf/ngf et recepteurs oligomeres solubles de la super-famille tnf/ngf

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EP0759984A1 true EP0759984A1 (fr) 1997-03-05
EP0759984A4 EP0759984A4 (fr) 2002-06-26

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WO (1) WO1995031544A1 (fr)

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US6355780B1 (en) 1995-02-22 2002-03-12 Yeda Research And Development Co. Ltd. Antibodies to the death domain motifs of regulatory proteins
US6747138B1 (en) 1995-04-03 2004-06-08 Regents Of The University Of Michigan Methods and compositions for regulating Fas-associated apoptosis
US7807783B1 (en) 1995-04-03 2010-10-05 The Regents Of The University Of Michigan Methods and compositions for regulating FAS-associated apoptosis
US6399327B1 (en) 1995-07-16 2002-06-04 Yeda Research And Development Co. Ltd. Modulators of the function of FAS receptors and other proteins
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US6593456B1 (en) 1996-11-06 2003-07-15 The Regents Of The University Of California Tumor necrosis factor receptor releasing enzyme
US6930084B1 (en) 1996-11-06 2005-08-16 The Regents Of The University Of California Treating arthritis with TNF receptor releasing enzyme
KR100533531B1 (ko) 1996-11-06 2005-12-06 더 리젠츠 오브 더 유니버시티 오브 캘리포니아 분리된 종양괴사인자 수용체 방출효소, 그 효소로 이루어지는 조성물 및 그것의 사용방법
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EP0759984A4 (fr) 2002-06-26
IL111125A0 (en) 1994-12-29
AU2546995A (en) 1995-12-05
KR100404737B1 (ko) 2004-05-03
CN1329512C (zh) 2007-08-01
AU703919B2 (en) 1999-04-01
CN1616661A (zh) 2005-05-18
KR970703419A (ko) 1997-07-03
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JP3966387B2 (ja) 2007-08-29
FI964509A0 (fi) 1996-11-08
CN1173033C (zh) 2004-10-27
CN1152937A (zh) 1997-06-25
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FI964509A (fi) 1997-01-09
NO964741L (no) 1997-01-09
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CA2189983A1 (fr) 1995-11-23
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