TNF ligands

Details TNF ligands TNF ligands

1995-06-07

2001-05-15

Gambel, Phillip (Department: 1644)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Blood proteins or globulins, e.g., proteoglycans, platelet...

C530S387100, C530S387900, C530S388100, C530S388200, C530S389100, C435S326000, C435S331000, C435S332000, C435S334000, C435S346000

Reexamination Certificate

active

06232446

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to ligands to Tumor Necrosis Factor receptors (TNF-Rs) which inhibit the effect of TNF but not its binding to the TNF-Rs, as well as to ligands interacting with other receptors of the TNF/NGF receptor family.
BACKGROUND OF THE INVENTION
Tumor necrosis factor (TNF) is a pleiotropic cytokine, produced by a number of cell types, mainly by activated macrophages. It is one of the principal mediators of the immune and inflammatory response. Interest in its function has greatly increased, recently, in view of evidence of the involvement of TNF in the pathogenesis of a wide range of disease states, including endotoxin shock, cerebral malaria and graft-versus-host reaction. Since many of the effects of TNF are deleterious to the organism, it is of great interest to find ways of blocking its action on host cells. An evident target for such intervention are the molecules to which TNF has to bind in order to exert its effects, namely the TNF-Rs. These molecules exist not only in cell-bound, but also in soluble forms, consisting of the cleaved extra-cellular domains of the intact receptors (see Nophar et al., EMBO Journal, 9(10):3269-78, 1990). The soluble receptors maintain the ability to bind TNF, and thus have the ability to block its function by competition with surface receptors.
Another method of TNF inhibition based on the principle of competing with cell-bound molecules, is the use of antibodies recognizing TNF receptors and blocking the ligand binding.
The cell surface TNF-Rs are expressed in almost all cells of the body. The various effects of TNF, the cytotoxic, growth-promoting and others, are all signalled by the TNF receptors upon the binding of TNF to them. Two forms of these receptors, which differ in molecular size: 55 and 75 kilodaltons, have been described, and well be called herein p55 and p75 TNF-R, respectively. It should be noted, however, that there exist publications which refer to these receptors also as p60 and p80.
The TNF-Rs belong to a family of receptors which are involved in other critical biological processes. Examples of these receptors are the low affinity NGF receptor, which plays an important role in the regulation of growth and differentiation of nerve cells. Several other receptors are involved in the regulation of lymphocyte growth, such as CDw40 and some others. Another member of the family is the FAS receptor also called APO, a receptor which is involved in signalling for apoptosis and which, based on a study with mice deficient in its function, seems to play an important role in the etiology of a lupus-like disease. Herein, this family of receptors is called “TNF/NGF receptor family”.
One of the most striking features of TNF compared to other cytokines, thought to contribute to the pathogenesis of several diseases, is its ability to elicit cell death. The cell-killing activity of TNF is thought to be induced by the p55 receptor. However, this p55 receptor activity can be assisted by the p75 receptor, through a yet unknown mechanism.
Parent application number 07/524,263 and European patent publication nos. 0,398,327 and 0,412,486 disclose antibodies to the soluble TNF-Rs. These antibodies were found to recognize the soluble TNF-Rs and to inhibit the binding of TNF to the TNF-Rs on the cell surface. Monovalent F(ab) fragments blocked the effect of TNF, while intact antibodies were observed to mimic the cytotoxic effect of TNF.
SUMMARY OF THE INVENTION
The present invention provides a ligand to a member of the TNF/NGF receptor family, which binds to the region of the C-terminal cysteine loop of such a receptor.
Preferably this region includes the amino acid sequence cys-163 to thr-179 in the p75 TNF-R or a corresponding region in another member of the TNF/NGF family.
Preferably, the receptor is the TNF-R, in particular the p75 TNF-R.
One such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 32, shown in
FIG. 11
(SEQ ID NO:7), and/or the amino acid sequence for the CDR region of the light chain of this antibody shown in
FIG. 12
(SEQ ID NO:11).
Another such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 70 (SEQ ID NO:5) shown in FIG.
11
.
Yet another such ligand includes the amino acid sequence for the CDR region of the heavy chain of monoclonal antibody no. 57 (SEQ ID NO:9), shown in FIG.
11
.
The above antibodies are called herein, for simplicity's sake, “group 32” antibodies.
In another aspect of the invention, the ligands comprise the scFv of a group 32 antibody.
The ligands may comprise e.g. proteins, peptides, immunoadhesins, antibodies or other organic compounds.
The proteins may comprise, for example, a fusion protein of the ligand with another protein, optionally linked by a peptide linker. Such a fusion protein can increase the retention time of the ligand in the body, and thus may even allow the ligand-protein complex to be employed as a latent agent or as a vaccine.
The term “proteins” includes muteins and fused proteins, their salts, functional derivatives and active fractions.
The peptides include peptide bond replacements and/or peptide mimetics, i.e. pseudopeptides, as known in the art (see e.g. Proceedings of the 20th European Peptide Symposium, ed. G. Jung, E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for oral, topical, nasal spray, ocular pulmonary, I.V. or subcutaneous delivery, depending on the particular treatment indicated. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable to enhance the stability, formulation, deliverability (e.g. slow release, prodrugs), or to improve the economy of production, as long as they do not adversely affect the biological activity of the peptide.
Besides substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation without adversely affecting activity. An example is given in the paper “Tritriated D-ala
1
-Peptide T Binding”, Smith C. S. et al., Drug Development Res. 15, pp. 371-379 (1988). Secondly, cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al. in Smith and Rivier (Eds.) “Peptides: Chemistry and Biology”, Escom, Leiden (1991), pp. 268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in conformationally restricted thymopentin-like compounds”, U.S. Pat. No. 4,457,489 (1985), Goldstein, G. et al. Thirdly, the introduction of ketomethylene, methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the interchange of the CO and NH moieties are likely to enhance both stability and potency. An example of this type is given in the paper “Biologically active retroinverso analogues of thymopentin”, Sisto A. et al in Rivier, J. E. and Marshall, G. R. (eds) “Peptides, Chemistry, Structure and Biology”, Escom, Leiden (1090), pp. 722-773).
The peptides of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, E: “Methoden der organischen Chemie”, Volume 15, Band 1+2, Synthese von Peptiden, thime Verlag, Stuttgart (1974), and Barrany, G.; Marrifield, R. B.: “The Peptides”, eds. E. Gross, J. Meienhofer, Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, F. Johansen, J. T., Carlsberg Res. Commun., Vol. 44, pp. 37-46 (1979), and Kullmann, W.: “Enzy

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